Reducing systemic regulatory T cell levels or activity for treatment of Alzheimer&#39;s disease

ABSTRACT

A pharmaceutical composition comprising an active agent that causes reduction of the level of systemic immunosuppression in an individual for use in treating a disease, disorder, condition or injury of the CNS that does not include the autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RRMS), is provided. The pharmaceutical composition is for administration by a dosage regimen comprising at least two courses of therapy, each course of therapy comprising in sequence a treatment session followed by an interval session.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation that claims priority pursuantto 35 U.S.C. §120 to U.S. patent application Ser. No. 14/797,894, filedon Jul. 13, 2015, a Continuation-in-Part application that claimspriority to International Application No. PCT/IL2015/050265, filed Mar.12, 2015, in which the United States is designated, and claims thebenefit of priority from U.S. Provisional Patent Application No.61/951,783 filed Mar. 12, 2014 and U.S. Provisional Patent ApplicationNo. 62/030,164 filed Jul. 29, 2014, the entire contents of each and allthese applications being hereby incorporated by reference herein intheir entirety as if fully disclosed herein.

FIELD OF THE INVENTION

The present invention relates in general to methods and compositions fortreating disease, disorder, condition or injury of the Central NervousSystem (CNS) by transiently reducing the level of systemicimmunosuppression in the circulation.

BACKGROUND OF THE INVENTION

Most central nervous system (CNS) pathologies share a commonneuroinflammatory component, which is part of disease progression, andcontributes to disease escalation. Among these pathologies isAlzheimer's disease (AD), an age-related neurodegenerative diseasecharacterized by progressive loss of memory and cognitive functions, inwhich accumulation of amyloid-beta (Aβ) peptide aggregates was suggestedto play a key role in the inflammatory cascade within the CNS,eventually leading to neuronal damage and tissue destruction (Akiyama etal, 2000; Hardy & Selkoe, 2002; Vom Berg et al, 2012). Despite thechronic neuroinflammatory response in neurodegenerative diseases,clinical and pre-clinical studies over the past decade, investigatingimmunosuppression-based therapies in neurodegenerative diseases, haveraised the question as to why anti-inflammatory drugs fall short(Breitner et al, 2009; Group et al, 2007; Wyss-Coray & Rogers, 2012). Weprovide a novel answer that overcomes the drawbacks of existingtherapies of AD and similar diseases and injuries of the CNS; thismethod is based on our unique understanding of the role of the differentcomponents of systemic and central immune system in CNS maintenance andrepair.

SUMMARY OF INVENTION

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an active agent that causes reduction of thelevel of systemic immunosuppression in an individual for use in treatinga disease, disorder, condition or injury of the CNS that does notinclude the autoimmune neuroinflammatory disease, relapsing-remittingmultiple sclerosis (RRMS), wherein said pharmaceutical composition isfor administration by a dosage regimen comprising at least two coursesof therapy, each course of therapy comprising in sequence a treatmentsession followed by an interval session of non-treatment.

In another aspect, the present invention provides a method for treatinga disease, disorder, condition or injury of the Central Nervous System(CNS) that does not include the autoimmune neuroinflammatory diseaserelapsing-remitting multiple sclerosis (RRMS), said method comprisingadministering to an individual in need thereof a pharmaceuticalcomposition comprising an active agent that causes reduction of thelevel of systemic immunosuppression according to the present invention,wherein said pharmaceutical composition is administered by a dosageregime comprising at least two courses of therapy, each course oftherapy comprising in sequence a treatment session followed by aninterval session of a non-treatment period.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-B depict the choroid plexus (CP) activity along diseaseprogression in the 5×FAD transgenic mouse model of AD (AD-Tg). (A) mRNAexpression levels for the genes icam1, vcam1, cxcl10 and ccl2, measuredby RT-qPCR, in CPs isolated from 1, 2, 4 and 8-month old AD-Tg mice,shown as fold-change compared to age-matched WT controls (n=6-8 pergroup; Student's t test for each time point). (B) Representativemicroscopic images of CPs of 8-month old AD-Tg mice and age-matched WTcontrols, immunostained for the epithelial tight junction moleculeClaudin-1, Hoechst nuclear staining, and the integrin lignad, ICAM-1(scale bar, 50 μm). In all panels, error bars represent mean±s.e.m.; *,P<0.05; **, P<0.01; ***, P<0.001.

FIGS. 2A-C show (A) Quantification of ICAM-1 immunoreactivity in humanpostmortem CP of young and aged non-CNS diseased, and AD patients (n=5per group; one-way ANOVA followed by Newman-Keuls post hoc analysis);(B) flow cytometry analysis for IFN-γ-expressing immune cells(intracellularly stained, and pre-gated on CD45) in CPs of 8-month oldAD-Tg mice and age-matched WT controls. Shaded histogram representsisotype control (n=4-6 per group; Student's t test); and (C) mRNAexpression levels of ifn-γ, measured by RT-qPCR, in CP tissues isolatedfrom 4- and 8-month old AD-Tg mice, compared to age-matched WT controls(n=5-8 per group; Student's t test for each time point). In all panels,error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01; ***, P<0.001.

FIGS. 3A-B depict (A) representative flow cytometry plots of CD4⁺Foxp3⁺splenocyte frequencies (pre-gated on TCRβ) in 8-month old AD-Tg and WTcontrol mice; and (B) quantitative analysis of splenocytes from 1, 2, 4and 8-month AD-Tg and WT control mice (n=6-8 per group; Student's t testfor each time point). In all panels, error bars represent mean±s.e.m.;*, P<0.05; **, P<0.01; ***, P<0.001.

FIG. 4 shows gating strategy and representative flow cytometry plots ofsplenocytes from AD-Tg/Foxp3-DTR^(+/−) mice, 1 day after the lastinjection of DTx. DTx was injected i.p. for 4 constitutive days,achieving ˜99% depletion of Foxp3⁺ cells.

FIGS. 5A-G show the effects of transient depletion of Tregs in AD-Tgmice. (A) AD-Tg/Foxp3-DTR⁺ (which express the DTR transgene) and anon-DTR-expressing AD-Tg littermate (AD-Tg/Foxp3-DTR⁻) control groupwere treated with DTx for 4 constitutive days. CP mRNA expression levelsfor the genes icam1, cxcl10 and ccl2, measured by RT-qPCR, in 6-monthold DTx-treated AD-Tg mice, 1 day after the last DTx injection (n=6-8per group; Student's t test). (B-D) Flow cytometry analysis of the brainparenchyma (excluding the choroid plexus, which was separately excised)of 6-month old DTx-treated AD-Tg mice and controls, 3 weeks followingthe last DIN injection. Quantitative flow cytometry analysis showingincreased numbers of CD11b^(high)/CD45^(high) mo-MΦ and CD4⁺ T cells(B), and representative flow cytometry plots (C) and quantitativeanalysis (D) of CD4⁺Foxp3⁺ Treg frequencies, in the brain parenchyma ofAD-Tg/Foxp3-DTR⁺ mice and AD-Tg/Foxp3-DTR⁻ controls treated with DTx(n=3-7 per group; Student's t test). (E) mRNA expression levels of foxp3and il10 in the brain parenchyma of 6-month old DTx-treated AD-TgAD-Tg/Foxp3-DTR⁺ and AD-Tg/Foxp3-DTR-contros, 3 weeks after the last DTxinjection (n=6-8 per group; Student's t test). (F) quantitative analysisof GFAP immunostaining, showing reduced astrogliosis in hippocampalsections from 6-month old DTx-treated AD-Tg/Foxp3-DTR⁺ andAD-Tg/Foxp3-DTR⁻ control mice. 3 weeks following the last DTx injection(scale bar, 50 μm; n=3-5 per group; Student's t test). (G) mRNAexpression levels of il-12p40 and tnf-α in the brain parenchyma, 3 weeksfollowing, the last DTx injection (n=6-8 per group; Student's t test).In all panels, error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIGS. 6A-E show the effect of transient depletion of Tregs on Aβ plaqueslearning/memory performance. (A) Representative microscopic images and(B) quantitative analysis of the brains of 5-month old DTx-treatedAD-Tg/Foxp3-DTR⁺ and AD-Tg/Foxp3-DTR⁻ control mice, 3 weeks after thelast DTx injection, immunostained for Aβ plaques and Hoechst nuclearstaining (scale bar, 250 μm). Mean Aβ plaque area and numbers in thehippocampal dentate gyrus (DG) and the 5^(th) layer of the cerebralcortex were quantified (in 6 μm brain slices; n=5-6 per group; Student'st test). FIGS. 6C-E) show Morris water maze (MWM) test performance of6-month old DTx-treated AD-Tg/Foxp3-DTR⁺ and control mice, 3 weeks afterthe last DTx injection. Following transient Treg depletion, AD-Tg miceshowed better spatial learning/memory performance in the (C)acquisition, (D) probe and (E) reversal phases of the MWM, relative toAD-Tg controls (n=7-9 per group; two-way repeated measures ANOVAfollowed by Bonferroni post-hoc analysis for individual paircomparisons; *, P<0.05 for overall acquisition, probe, and reversal). Inall panels, error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01;***, P<0.001.

FIG. 7 shows mRNA expression levels of ifn-γ, measured by RT-qPCR, inCPs isolated from 6- and 12-month old APP/PS1 AD-Tg mice (a mouse modelfor Alzheimer's disease (see Materials and Methods)), compared toage-matched WT controls (n=5-8 per group; Student's t test). Error barsrepresent mean±s.e.m.; *, P<0.05.

FIGS. 8A-I show the therapeutic effect of administration of weeklyGlatiramer acetate (GA) in AD-Tg mice. (A) Schematic representation ofweekly-GA treatment regimen. Mice (5-month old) were s.c. injected withGA (100 μg), twice during the first week (on day 1 and 4), and onceevery week thereafter, for an overall period of 4 weeks. The mice wereexamined for cognitive performance, 1 week (MWM), 1 month (RAWM) and 2months (RAWM, using different experimental spatial settings) after thelast injection, and for hippocampal inflammation. FIGS. 8B-D show mRNAexpression levels of genes in the hippocampus of untreated AD-Tg mice,and AD-Tg mice treated with weekly-GA, at the age of 6 m, showing (B)reduced expression of pro-inflammatory cytokines such as TNF-α, IL-1βand IL-12p40, (C) elevation of the anti-inflammatory cytokines IL-10 andTGF-β, and of (D) the neurotropic factors, IGF-1 and BDNF, in weekly-GAtreated mice (n=6-8 per group; Student's t test). In FIGS. 8E-G, AD-Tgmice (5 months old) were treated with either weekly-GA or with vehicle(PBS), and compared to age-matched WT littermates in the MWM task at theage of 6 m. Treated mice showed better spatial learning/memoryperformance in the acquisition (E), probe (F) and reversal (G) phases ofthe MWM, relative to controls (n=6-9 per group; two-way repeatedmeasures ANOVA followed by Bonferroni post-hoc for individual paircomparisons; WT mice, black circles; AD-Tg controls, white circles;treated AD-Tg, grey circles). FIGS. 8H-I show cognitive performance ofthe same mice in the RAWM task, 1 month (H) or 2 months (I) followingthe last GA injection (n=6-9 per group; two-way repeated measures ANOVAfollowed by Bonferroni post-hoc for individual pair comparisons). Dataare representative of at least three independent experiments. In allpanels, error bars represent mean±s.e.m.; *, P<0.05; **, P<0.01; ***,P<0.001.

FIGS. 9A-H show further therapeutic effects of administration ofweekly-GA in AD-Tg mice. A-B shows 5×FAD AD-Tg mice that were treatedwith either weekly-GA, or vehicle (PBS), and were examined at the end ofthe 1^(st) week of the administration regimen (after a total of two GAinjections). Flow cytometry analysis for CD4⁺Foxp3⁺ splenocytefrequencies (A), and CP IFN-γ-expressing immune cells (B;intracellularly stained and pre-gated on CD45), in treated 6-month oldAD-Tg mice, compared to age-matched WT controls (n=4-6 per group;one-way ANOVA followed by Newman-Keuls post hoc analysis). (C) mRNAexpression levels for the genes icam1, cxcl10 and ccl2, measured byRT-qPCR, in CPs of 4-month old AD-Tg mice, treated with either weekly-GAor vehicle, and examined either at the end of the 1^(st) or 4^(th) weekof the weekly-GA regimen (n=6-8 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis). FIGS. 9D-E show representative imagesof brain sections from 6-month old AD-Tg/CX₃CR1^(GFP/+) BM chimerasfollowing weekly-GA. CX₃CR1^(GFP) cells were localized at the CP of thethird ventricle (3V; i), the adjacent ventricular spaces (ii), and theCP of the lateral ventricles (LV; iii) in AD-Tg mice treated withweekly-GA (D; scale bar, 25 μm). Representative orthogonal projectionsof confocal z-axis stacks, showing co-localization of GFP⁺ cells withthe myeloid marker, CD68, in the CP of 7-month old AD-Tg/CX₃CR1^(GFP/+)mice treated with weekly-GA, but not in control PBS-treatedAD-Tg/CX₃CR1^(GFP/+) mice (E; scale bar, 25 μm). (F) CX₃CR1^(GFP) cellsare co-localized with the myeloid marker IBA-1 in brains of GA-treatedAD-Tg/CX₃CR1^(GFP/+) mice in the vicinity of Aβ plaques, andco-expressing the myeloid marker, IBA-1 (scale bar, 25 μm). FIGS. 9G-Hshow representative flow cytometry plots of cells isolated from thehippocampus of 4-month old WT, untreated AD-Tg, and AD-Tg mice, on the2^(nd) week of the weekly-GA regimen. CD11b^(high)/CD45^(high) mo-MΦwere gated (G) and quantified (H; n=4-5 per group; one-way ANOVAfollowed by Newman-Keuls post hoc analysis). In all panels, error barsrepresent mean±s.e.m.; *, P<0.05; **, P<0.01; ***, P<0.001.

FIGS. 10A-H depict the therapeutic effect of administration of a p300inhibitor (C646) in AD-Tg mice. In FIGS. 10A-B, aged mice (18 months)were treated with either p300i or vehicle (DMSO) for a period of 1 week,and examined a day after cessation of treatment. Representative flowcytometry plots showing elevation in the frequencies of CD4⁺ T cellsexpressing IFN-γ in the spleen (A), and IFN-γ-expressing immune cellnumbers in the CP (B), following p300i treatment. FIGS. 10C-E showrepresentative microscopic images (C), and quantitative analysis, of Aβplaque burden in the brains of 10-month old AD-Tg mice, which receivedeither p300i or vehicle (DMSO) for a period of 1 week, and weresubsequently examined after 3 additional weeks. Brains wereimmunostained for Aβ plaques and by Hoechst nuclear staining (n=5 pergroup; Scale bar, 250 μm). Mean Aβ plaque area and plaque numbers werequantified in the hippocampal DG (D) and the 5^(th) layer of thecerebral cortex (E) (in 6 μm brain slices; n=5-6 per group; Student's ttest). (F) Schematic representation of the p300i treatment (or DMSO asvehicle) administration regimen to the different groups of AD-Tg mice atthe age of 7 months, in either 1 or 2 sessions. FIGS. 10G-H show thechange mean of Aβ plaque percentage coverage of the cerebral cortex(5^(th) layer) (G), and the change in mean cerebral soluble Aβ₁₋₄₀ andAβ₁₋₄₂ protein levels (H), relative to the untreated AD-Tg group (Aβ₁₋₄₀and Aβ₁₋₄₂ mean level in untreated group, 90.5±11.2 and 63.8±6.8 μg/mgtotal portion, respectively; n=5-6 per group; one-way ANOVA followed byNewman-Keuls post hoc analysis). In all panels, error bars representmean±s.e.m.; *, P<0.05; **, P<0.01; ***, P<0.001.

FIGS. 11 A-D show that PD-1 blockade augments IFN-γ-dependent choroidplexus activity in AD-Tg mice. 10-month old AD-Tg mice were i.p.injected on day 1 and day 4 with 250 ug of either anti-PD-1 or controlIgG, and examined at days 7-10 for the effect on the systemic immuneresponse and CP activity. (A-B) Representative flow cytometry plots (A),and quantitative analysis (B), of CD4⁺IFN-γ⁺ splenocyte frequencies(intracellularly stained and pre-gated on CD45 and TCR-β), in αPD-1 orIgG treated AD-Tg mice, and untreated AD-Tg and WT controls (n=4-6 pergroup; one-way ANOVA followed by Newman-Keuls post hoc analysis; **,P<0.01 between the indicted treated groups; error bars representmean±s.e.m.). (C) mRNA expression levels of ifn-g, measured by RT-qPCRin the CP of AD-Tg mice treated with anti-PD-1 when compared to IgGtreated and untreated AD-Tg controls (D) GO annotation terms enriched inRNA-Seq in CPs of the same mice (n=3-5 per group; one-way ANOVA followedby Newman-Keuls post hoc analysis; *, P<0.05) (gray scale corresponds tonegative log-base 10 of P-value).

FIGS. 12A-B show that PD-1 blockade mitigates cognitive decline in AD-Tgmice. 10-month old AD-Tg mice were i.p. injected on day 1 and day 4 with250 ug of either anti-PD-1 or control IgG, and examined 1 or 2 monthslater for the effect on pathology. (A-B) Scheme of the experimentaldesign. Single arrows indicate time points of treatment, and doublearrows indicate time points of cognitive testing. Cognitive performanceof anti-PD-1 and IgG treated mice, compared to age-matched WT anduntreated AD-Tg mice, assessed by the average number of errors per dayin the RAWM learning and memory task (n=6-8 per group; two-way repeatedmeasures ANOVA followed by Bonferroni post-hoc for individual paircomparisons). (A) Performance of AD-Tg mice in the RAWM after 1treatment session with anti-PD-1 or IgG control. (B) Effect of singleanti-PD-1 treatment session, or 2 sessions with a 1 month interval onperformance.

FIGS. 13A-D depict representative microscopic images showing that PD-1blockade mitigates AD pathology (A), and quantitative analyses (B, C,D), of Aβ plaque burden and astrogliosis in the brains of AD-Tg mice,which were treated at the age of 10-months with either anti-PD-1 (in 1or 2 sessions, as depicted in FIG. 12a-b ) or IgG control, andsubsequently examined at the age of 12 months. Brains were immunostainedfor Aβ plaques (in red), GFAP (marking astrogliosis, in green), and byHoechst nuclear staining (n=4-5 per group; Scale bar, 50 μm). Mean Aβplaque area and plaque numbers were quantified in the hippocampaldentate gyrus (DG) and the 5^(th) layer of the cerebral cortex, and GFAPimmunoreactivity was measured in the hippocampus (in 6 μm brain slices;n=5-6 per group; Student's t test). In all panels, error bars representmean±s.e.m.; *, P<0.05; **, P<0.01; ***, P<0.001.

DETAILED DESCRIPTION

Immune checkpoint mechanisms, which include cell-intrinsicdownregulation of activated T cell responsiveness and effector functionby inhibitory receptors, maintain systemic immune homeostasis andautoimmune tolerance (Joller et al, 2012; Pardoll, 2012). In recentyears, blockade of these immune checkpoints, such as the programmeddeath-1 (PD-1) pathway (Francisco et al, 2010), has demonstrated notableanti-tumor efficacy, highlighting the potential of unleashing the powerof the immune system in fighting various malignancies (Postow et al,2015). Similarly, the findings disclosed herein (Example 5) provide thefirst evidence of the therapeutic potential of immune checkpointblockade in a neurodegenerative disease, such as AD. Though breakingtolerance is now widely accepted in cancer immunotherapy (Lesokhin etal, 2015; Mellman et al, 2011; Schreiber et al, 2011), this approach intreating chronic neurodegenerative diseases has been overlooked. It isdisclosed herein that in AD-Tg mice, breaking immune tolerance by PD-1blockade resulted in a systemic IFN-γ-associated response, as wasdescribed in cancer immunotherapy (Lesokhin et al, 2015; Peng et al,2012). The inventors also found that PD-1 blockade had a tissue-specificeffect of IFN-γ response at the CP. Such response was previously shownby the inventors to be essential for driving CNS repair processesthrough an immunological mechanism involving enhanced CNS immunesurveillance in mouse models of acute (Kunis et al, 2013; Shechter etal, 2013) and chronic (Baruch et al, 2015; Kunis et al, 2015)neurodegeneration. Testing the effect on cerebral Aβ plaque pathology,it is disclosed herein that a single treatment session utilizing PD-1blockade led to a significant reduction in plaque burden that lasted forat least 2 months, the last time point tested. Notably, an additionaltreatment session with anti-PD-1, a month after the initial treatment,was necessary to maintain the effect on cognitive performance. Thesefindings suggest that for long-term efficacy, repeated treatmentsessions should be considered, at intervals to be determined in furtherstudies.

Relief of systemic immune suppression may be achieved by means otherthan release of immune checkpoints, for example by a decrease insystemic regulatory T cells, or attenuating their activity. Thus, it hasfurther been found in accordance with the present invention that ashort-term transient depletion of Foxp3⁺ regulatory T cells (Tregs) in amouse model of Alzheimer's disease (AD-Tg mice) results in improvedrecruitment of leukocytes to the CNS through the brain's choroid plexus,elevated numbers of CNS-infiltrating anti-inflammatory monocyte-derivedmacrophages mo-MΦ and CD4⁺ T cells, and a marked enrichment ofFoxp3⁺Tregs that accumulates within the brain. Furthermore, thelong-term effect of a single session of treatment lead to a reduction inhippocampal gliosis and reduced mRNA expression levels ofpro-inflammatory cytokines within the brain. Importantly, the effect ondisease pathology includes reduced cerebral amyloid beta (Aβ) plaqueburden in the hippocampal dentate gyrus, and the cerebral cortex (5^(th)layer), two brain regions exhibiting robust Aβ plaque pathology in theAD-Tg mice. Most importantly, the short-term transient depletion ofTregs is followed by a dramatic improvement in spatial learning andmemory, reaching cognitive performance similar to that of wild type mice(Examples 2 and 3). Taken together, these findings demonstrate that ashort session of Treg depletion, followed by a period of nointervention, results in transiently breaking Treg-mediated systemicimmune suppression in AD-Tg mice, which enables recruitment ofinflammation-resolving cells, mo-MΦ and Tregs, to the brain, and lead toresolution of the neuroinflammatory response, clearance of Aβ, andreversal of cognitive decline.

These findings strongly argue against the common wisdom in this field ofresearch, according to which increasing systemic immune suppressionwould result in mitigation of the neuroinflammatory response. On thecontrary, our findings show that boosting of the systemic response, by ashort-term, brief and transient, reduction in systemic Treg-mediatedsuppression or release of restraints on the immune system in the form ofimmune checkpoints, is needed in order to achieve inflammation-resolvingimmune cell accumulation, including Tregs themselves, within the brain,thus fighting off AD pathology.

The specificity of the inventors approach presented herein has beensubstantiated by using several independent experimental paradigms, asdetailed below. Briefly, first the inventors used an immunomodulatorycompound in two different administration regimens that led to oppositeeffects on peripheral Treg levels, on CP activation, and on diseasepathology; a daily administration regimen that augments peripheral Treglevels (Weber et al, 2007), and a weekly administration regimen, whichthey found to reduce peripheral Treg levels (Example 3). The inventorsalso provide a direct functional linkage between peripheral Treg levelsand disease pathology when demonstrating in AD-Tg mice, by eithertransient in vivo genetic depletion of Tregs (Example 2), or bypharmacologic inhibition of their Foxp3 function (Examples 3 and 4),that these manipulations result in activation of the CP for facilitatingleukocyte trafficking to the CNS, inflammation-resolving immune cellaccumulation at sites of pathology, clearance of cerebral Aβ plaques,and skewing of the immunological milieu of the brain parenchyma towardsthe resolution of inflammation.

It has further been found in accordance with the present invention thatinfrequent administration of a universal antigen, Copolymer-1, for alimited period of time (representing one session of treatment) reducesTreg-mediated systemic immune suppression, and improves selectiveinfiltration of leukocytes into the CNS by increasing the brain'schoroid plexus gateway activity, leading to dramatic beneficial effectin Alzheimer's disease pathology (Example 3), while daily administrationof Copolymer 1, that enhance Treg immune suppression (Hong et al, 2005;Weber et al, 2007), showed no beneficial effect, or even some modestdetrimental effect, on disease pathology (Example 5 inPCT/IL2015/050265). The inventors of the present invention further showherein that direct interference with Foxp3 Treg activity, either byinhibition of p300 with a specific small molecule inhibitor (p300i), orinteraction with the PD-1 receptor by an anti-PD-1 antibody, improveschoroid plexus gateway activity in AD-Tg mice, and mitigates Alzheimer'sdisease pathology (Example 4).

Importantly, each of these examples provided by the inventors,demonstrate a different intervention which causes short term reductionin systemic immune suppression: Copolymer-1 acts as an immunomodulatorycompound, p300i as a small molecule which decreases Foxp3 acetylationand Treg function, and anti-PD-1 is used as a neutralizing antibody forPD-1 expressed on Tregs and as a immune checkpoint blocker. Thesetherapeutic approaches were used for a short session of treatment thattransiently augmented immune response in the periphery, mainly byelevation of peripheral IFN-γ levels and IFN-γ-producing cells, thusactivating the brain's choroid plexus allowing selective infiltration ofT cells and monocytes into the CNS, and homing of these cells to sitesof pathology and neuroinflammation. It was also found herein thatrepeated sessions of treatment interrupted by interval sessions ofnon-treatment dramatically improve the efficacy of the treatmentrelative to a single session of treatment (Example 4). The followingtime interval of non-treatment allowed transient augmentation in Treglevels and activities within the brain, facilitating the resolution ofneuroinflammation, and inducing environmental conditions in favor of CNShealing and repair, subsequently leading to tissue recovery. In each ofthese cases the effect on brain pathology was robust, involving theresolution of the neuroinflammatory response, amyloid beta plaqueclearance from AD mice brains, and reversal of cognitive decline. Thespecificity of the current approach has further been substantiated usinga genetic model of transient depletion of Foxp3⁺ regulatory T cells, intransgenic mouse model of AD (Example 2).

Thus, it has been found in accordance with the present invention thatsystemic immunosuppression interferes with ability to fight off ADpathology, acting at least in part, by inhibiting IFN-γ-dependentactivation of the CP, needed for orchestrating recruitment ofinflammation-resolving leukocytes to the CNS (Schwartz & Baruch, 2014b).Systemic Tregs are crucial for maintenance of autoimmune homeostasis andprotection from autoimmune diseases (Kim et al, 2007). However, ourfindings suggest that under neurodegenerative conditions, when areparative immune response is needed in the brain, the ability to mountthis response is interfered with by systemic Tregs or some other factorthat can be overcome by immune checkpoint blockade. Neverthelessaccording to our results, Tregs are needed within the brain, home tosites of neuropathology, and perform locally an anti-inflammatoryactivity. The present invention represents a unique and unexpectedsolution for the apparent contradictory needs in fighting offprogressive neuronal death as in AD; transiently reducing/inhibitingTregs in the circulation on behalf of increasing Tregs in the diseasedbrain. Hence, a short-term and transient reduction in peripheral immunesuppression, which allows the recruitment of anti-inflammatory cells,including Tregs and mo-MΦ, to sites of cerebral plaques, leads to along-term effect on pathology. Notably, however, a transient reductionof systemic Treg levels or activities or transient immune checkpointblockade may contribute to disease mitigation via additional mechanisms,including supporting a CNS-specific protective autoimmune response(Schwartz & Baruch, 2014a), or augmenting the levels of circulatingmonocytes that play a role in clearance of vascular Aβ (Michaud et al,2013).

Though neurodegenerative diseases of different etiology, share a commonlocal neuroinflammatory component, our results strongly argue againstsimplistic characterization of all CNS pathologies as diseases thatwould uniformly benefit from systemic anti-inflammatory therapy. Thus,while autoimmune inflammatory brain pathologies, such asRelapsing-Remitting Multiple Sclerosis (RRMS), benefit from continuoussystemic administration of anti-inflammatory and immune-suppressivedrugs to achieve long lasting peripheral immune suppression, it willeither be ineffective or detrimentally affect (Example 5 inPCT/IL2015/050265) pathology in chronic neurodegenerative diseases suchas in the case of AD, primary progressive multiple sclerosis (PP-MS) andsecondary-progressive multiple sclerosis (SP-MS). Moreover, our findingsshed light on the misperception regarding the role of systemic vs.tissue-associated Tregs in these pathologies (He & Balling, 2013). Sincethe immune-brain axis is part of life-long brain plasticity (Baruch etal, 2014), and neurodegenerative diseases are predominantly age-related,our present findings also point to a more general phenomenon, in whichsystemic immune suppression interferes with brain function. Accordingly,short-term periodic courses of reducing systemic immune suppression mayrepresent a therapeutic or even preventive approach, applicable to awide range of brain pathologies, including AD and age-associateddementia.

Importantly, the inventors approach and findings present herein in ADmouse models, do not directly target any disease-specific factor in AD,such as amyloid beta or tau pathology, but rather demonstrate a novelapproach which is expected to be clinically applicable in a wide rangeof CNS pathologies—transient reduction of systemic Treg-mediated immunesuppression or release of systemic immune suppression by blockade ofimmune checkpoints—in order to augment recruitment ofinflammation-resolving immune cells to sites of pathology within theCNS.

In view of the unexpected results described above, the present inventionprovides a method for treating a disease, disorder, condition or injuryof the Central Nervous System (CNS) that does not include the autoimmuneneuroinflammatory disease relapsing-remitting multiple sclerosis (RRMS),said method comprising administering to an individual in need thereof anactive agent that causes reduction of the level of systemicimmunosuppression, wherein said active agent is administered by a dosageregime comprising at least two courses of therapy, each course oftherapy comprising in sequence a treatment session followed by aninterval.

In another aspect, the present invention is directed to an active agentthat causes reduction of the level of systemic immunosuppression in anindividual, or a pharmaceutical composition comprising the active agent,for use in treating a disease, disorder, condition or injury of the CNSthat does not include the autoimmune neuroinflammatory disease,relapsing-remitting multiple sclerosis (RRMS), wherein saidpharmaceutical composition is for administration by a dosage regimencomprising at least two courses of therapy, each course of therapycomprising in sequence a treatment session followed by an intervalsession of non-treatment.

In certain embodiments, the dosage regimen is calibrated such that thelevel of systemic immunosuppression is transiently reduced.

The term “treating” as used herein refers to means of obtaining adesired physiological effect. The effect may be therapeutic in terms ofpartially or completely curing a disease and/or symptoms attributed tothe disease. The term refers to inhibiting the disease, i.e. arrestingor slowing its development; or ameliorating the disease, i.e. causingregression of the disease.

The term “non-treatment session” is used interchangeably herein with theterm “period of no treatment” and refers to a session during which noactive agent is administered to the individual being treated.

The term “systemic presence” of regulatory or effector T cells as usedherein refers to the presence of the regulatory or effector T cells (asmeasured by their level or activity) in the circulating immune system,i.e. the blood, spleen and lymph nodes. It is a well-known fact in thefield of immunology that the cell population profile in the spleen isreflected in the cell population profile in the blood (Zhao et al,2007).

The present treatment is applicable to both patients that show elevationof systemic immune suppression, as well as to patients that do not showsuch an elevation. Sometimes the individual in need for the treatmentaccording to the present invention has a certain level of peripheralimmunosuppression, which is reflected by elevated frequencies or numbersof Tregs in the circulation, and/or their enhanced functional activityand/or a decrease in IFNγ-producing leukocytes and/or decreasedproliferation of leukocytes in response to stimulation. The elevation offrequencies or numbers of Tregs can be in total numbers or as percentageof the total CD4 cells. For example, it has been found in accordancewith the present invention that an animal model of Alzheimer's diseasehas higher frequencies of Foxp3 out of CD4 cells as compared withwild-type mice. However, even if the levels of systemic Treg cells isnot elevated, their functional activity is not enhanced, the level ofIFNγ-producing leukocytes is not reduced or the proliferation ofleukocytes in response to stimulation is not decreased, in saidindividual, the method of the present invention that reduces the levelor activity of systemic immunosuppression is effective in treatingdisease, disorder, condition or injury of the CNS that does not includethe autoimmune neuroinflammatory disease RRMS. Importantly, saidsystemic immune suppression can also involve additional immune celltypes except of Tregs, such as myeloid-derived suppressor cells (MDSCs)(Gabrilovich & Nagaraj, 2009).

The level of systemic immunosuppression may be detected by variousmethods that are well known to those of ordinary skill in the art. Forexample, the level of Tregs may be measured by flow cytometry analysisof peripheral blood mononuclear cells or T lymphocytes, immunostainedeither for cellular surface markers or nuclear intracellular markers ofTreg (Chen & Oppenheim, 2011), CD45, TCR-β, or CD4 markers oflymphocytes, and measuring the amount of antibody specifically bound tothe cells. The functional activity of Tregs may be measured by variousassays; For example the thymidine incorporation assay is being commonlyused, in which suppression of anti-CD3 mAb stimulated proliferation ofCD4⁺CD25⁻ T cells (conventional T cells) is measured by [³H]thymidineincorporation or by using CFSE (5-(and 6)-carboxyfluorescein diacetatesuccinimidyl ester, which is capable of entering the cells; celldivision is measured as successive halving of the fluorescence intensityof CFSE). The number of IFNγ-producing leukocytes or their activity ortheir proliferation capacity can easily be assessed by a skilled artisanusing methods known in the art; For example, the level of IFNγ-producingleukocytes may be measured by flow cytometry analysis of peripheralblood mononuclear cells, following short ex-vivo stimulation andgolgi-stop, and immunostaining by IFNγ intracellular staining (usinge.g., BD Biosciences Cytofix/Cytoperm™ fixation/permeabilization kit),by collecting the condition media of these cells and quantifying thelevel of secreted cytokines using ELISA, or by comparing the ratio ofdifferent cytokines in the condition media, for example 1L2/IL10,IL2/IL4, INFγ/TGFβ, etc. The levels of MDSCs in the human peripheralblood easily can be assessed by a skilled artisan, for example by usingflow cytometry analysis of frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+,DR⁻/LIN⁻/CD33+ and DR(-/low)/CD14+ cells, as described (Kotsakis et al,2012).

In humans, the peripheral/systemic immunosuppression may be consideredelevated when the total number of Tregs in the circulation is higherthan 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more than in ahealthy control population, the percentage of Treg cells out of thetotal CD4+ cells is elevated by 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more than in a healthy control population, or the functionalactivity of Tregs is elevated by 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% or more than in a healthy control population. Alternatively, theperipheral/systemic immunosuppression may be considered elevated whenthe level of IFNγ-producing leukocytes or their activity is reducedrelative to that of a healthy control population by 10, 20, 30, 40, 50,60, 70, 80, 90 or 100%; or the proliferation of leukocytes in responseto stimulation is reduced relative to that of a healthy controlpopulation by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

An agent may be considered an agent that causes reduction of the levelof systemic immunosuppression when, upon administration of the agent toan individual, the total number of Tregs in the circulation of thisindividual is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% ascompared with the level before administration of the agent, thepercentage of Treg cells out of the total CD4+ cells drops by 10, 20,30, 40, 50, 60, 70, 80, 90 or 100% relative to that of a healthy controlpopulation or the functional activity of Tregs is reduced by 10, 20, 30,40, 50, 60, 70, 80, 90 or 100% as compared with the level beforeadministration of the agent. Alternatively, an agent may be consideredan agent that causes reduction of the level of systemicimmunosuppression when, upon administration of the agent to anindividual, the total number of IFNγ-producing leukocytes or theiractivity is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% ormore; or the proliferation of leukocytes in response to stimulation isincreased relative to that of a healthy control population by 10, 20,30, 40, 50, 60, 70, 80, 90 or 100% or more. In certain embodiments, theactive agent causes reduction of the level of systemic immunosuppressionby release of a restraint imposed on the immune system by one or moreimmune checkpoints, for example by blockade of the one or more immunecheckpoints.

In certain embodiments, the reduction of the level of systemicimmunosuppression is associated with an increase in systemic presence oractivity of IFNγ-producing leukocytes.

In certain embodiments, the active agent causes reduction of the levelof systemic immunosuppression and thereby an increase in the systemicpresence or activity of effector T cells.

The checkpoints that may be manipulated to release the systemicimmunosuppression are referred to herein as a pair of an immune checkpoint receptor and its native ligand, except when one partner of thepair is unknown, in which case only the known partner is referred to.For example, PD1, which has two known ligands is referred to herein as“PD1-PDL1” or “PD1-PDL2”, while B7H3, the ligand of which has not yetbeen identified, is referred to simply by “B7H3”.

The checkpoints that may be manipulated to release the systemicimmunosuppression in accordance with the present invention may beselected from the group consisting of PD1-PDL1, PD1-PDL2, CD28-CD80,CD28-CD86, CTLA4-CD80, CTLA4-CD86, ICOS-B7RP1, B7H3, B7H4, B7H7,B7-CD28-like molecule, BTLA-HVEM, KIR-MHC class I or II, LAG3-MHC classI or II, CD137-CD137L, OX40-OX40L, CD27-CD70, CD40L-CD40, TIM3-GAL9,V-domain Ig suppressor of T cell activation (VISTA), STimulator ofINterferon Genes (STING), T cell immunoglobulin and immunoreceptortyrosine-based inhibitory motif domain (TIGIT), A2aR-Adenosine andindoleamine-2,3-dioxygenase (IDO)-L-tryptophan.

Agents capable of blocking immune checkpoints are known in the art(Colombo & Piconese, 2007) and these agents can be used in accordancewith the present invention. Each one of the cited publications below,and Pardoll, 2012, is incorporated by reference as if fully disclosedherein.

In certain embodiments, the active agent that may be used according tothe present invention may be selected from the group consisting of:

(i) an antibody selected from the group consisting of: (a) anti-PD-1,(b) anti-PD-L1, (c) anti-PD-L2 (Coyne & Gulley, 2014; Duraiswamy et al,2014; Zeng et al, 2013); (d) anti-CTLA-4 (Simpson et al, 2013; Terme etal, 2012); (e) anti-CD80; (f) anti-CD86; (g) anti-B7RP1; (h) anti-B7-H3;(i) anti-B7-H4; (j) anti-BTLA; (k) anti-HVEM; (l) anti-CD137; (m)anti-CD137L; (n) anti-CD-27; (o) anti-CD70; (p) anti-CD40; (q)anti-CD40L; (r) anti-OX40 (Voo et al, 2013); (s) anti-OX40L; (t)anti-TIM-3/Galectin9; (u) anti-killer-cell immunoglobulin-like receptor(Ju et al, 2014); (v) anti-LAG-3; and (w) any combination of (a) to (v);

(ii) any combination of (a) to (v) in combination with an adjuvant, suchas anti-CTLA-4 antibody in combination with anti OX40 antibody and aTLR9 ligand such as CpG (Marabelle et al, 2013);

(iii) a small molecule selected from the group consisting of: (a) a p300inhibitor (Liu et al, 2013), such as gemcitabine (low dose) (Shevchenkoet al, 2013), or C646 or analogs thereof, i.e. a compound of the formulaI:

wherein

R₁ is selected from H, —CO₂R₆, —CONR₆R₇, —SO₃H, or —SO₂NR₆R₇;

R₂ is selected from H, —CO₂R₆, or halogen, preferably Cl;

R₃ is selected from halogen, preferably F, —NO₂, —CN, —CO₂R₆, preferablyCO₂CH₃ or CO₂CH₂CH₃, or —CH₂OH;

R₄ and R₅ each independently is H or —C₁-C₆ alkyl, preferably methyl;

R₆ is H or —C₁-C₆ alkyl, preferably H, methyl or ethyl; and

R₇ is H or —C₁-C₆ alkyl, preferably H or methyl (see (Bowers et al,2010));

(b) Sunitinib (Terme et al, 2012); (c) Polyoxometalate-1 (POM-1)(Ghiringhelli et al, 2012); (d) α,β-methyleneadenosine 5′-diphosphate(APCP) (Ghiringhelli et al, 2012); (e) arsenic trioxide (As₂O₃)(Thomas-Schoemann et al, 2012); (f) GX15-070 (Obatoclax) (Kim et al,2014); (g) a retinoic acid antagonist such as Ro 41-5253 (a syntheticretinoid and selective small molecule antagonist) (Galvin et al, 2013)or LE-135 (Bai et al, 2009); (h) an SIRPα (CD47) antagonist, such asCV1-hIgG4 (SIRPα variant) as sole agent or in combination with anti-CD47antibody (Weiskopf et al, 2013); (i) a CCR4 antagonist, such asAF399/420/18025 as sole agent or in combination with anti-CCR4 antibody(Pere et al, 2011); (j) an adenosine receptor antagonist; (k) anadenosine A1 receptor antagonist; an adenosine A2a receptor; (m) anadenosine A2b receptor antagonist; (n) an A3 receptor antagonist; (o) anantagonist of indoleamine-2,3-dioxygenase; and (p) an HIF-1 regulator;

(iv) any combination of (iii) (a-p) and (i) (a-v);

(v) a protein selected from the group consisting of: (a) Neem leafglycoprotein (NLGP; (Roy et al, 2013)); and (b) sCTLA-4 (soluble isoformof CTLA-4) (Ward et al, 2013);

(vi) a silencing molecule selected from the group consisting of miR-126antisense (Qin et al, 2013) and anti-galectin-1 (Gal-1; (Dalotto-Morenoet al, 2013));

(vii) OK-432 (lyophilized preparation of Streptococcus pyogenes)(Hirayama et al, 2013);

(viii) a combination of IL-12 and anti-CTLA-4;

(ix) an antibiotic agent such as vancomycin (Brestoff & Artis, 2013;Smith et al, 2013); or

(x) any combination of (i) to (viii).

In certain embodiments, the agent is an anti-PD-1 antibody, i.e. anantibody specific for PD-1.

Many anti-PD-1 antibodies are known in the art. For example, theanti-PD-1 antibody used in accordance with the present invention may beselected from those disclosed in Ohaegbulam et al. (Ohaegbulam et al,2015), the entire contents of which being hereby incorporated herein byreference, i.e. CT-011 (pidilizumab; Humanized IgG1; Curetech), MK-3475(lambrolizumab, pembrolizumab; Humanized IgG4; Merck), BMS-936558(nivolumab; Human IgG4; Bristol-Myers Squibb), AMP-224 (PD-L2 IgG2afusion protein; AstraZeneca), BMS-936559 (Human IgG4; Bristol-MyersSquibb), MEDI4736 (Humanized IgG; AstraZeneca), MPDL3280A (Human IgG;Genentech), MSB0010718C (Human IgG1; Merck-Serono); or the antibody usedin accordance with the present invention may be MEDI0680 (AMP-514;AstraZeneca) a humanized IgG4 mAb.

In certain embodiments, the CT-011 antibody may be administered to ahuman at a dosage of 0.2-6 mg/kg or between 1.5-6 mg/kg; the MK-3475antibody may be administered to a human at a dosage of 1-10 mg/kg;BMS-936558 may be administered to a human at a dosage of 0.3-20 mg/kg,0.3-10 mg/kg, 1-10 mg/kg or at 1 or 3 mg/kg; BMS-936559 may beadministered to a human at a dosage of 0.3-10 mg/kg; MPDL3280A may beadministered to a human at a dosage of 1-20 mg/kg; MEDI4736 may beadministered to a human at a dosage of 0.1-15 mg/kg; and MSB0010718C maybe administered to a human at a dosage of 1-20 mg/kg.

The anti-CTLA4 antibody may be Tremelimumab (Pfizer), a fully human IgG2monoclonal antibody; or ipilimumab, a fully human human IgG1 monoclonalantibody.

The anti-killer-cell immunoglobulin-like receptors (KIR) antibody may beLirilumab (BMS-986015; developed by Innate Pharma and licenced toBristol-Myers Squibb), a fully human monoclonal antibody.

The anti-LAG-3 antibody is directed against lymphocyte activationgene-3. One such antibody that may be used according to the presentinvention is the monoclonal antibody BMS-986016 (pembrolizumab;Humanized IgG4; Merck).

TABLE 1* C646 C375 C146

*Based on Bowers et al. (2010)

In certain embodiments, combinations of antibodies may be used such asbut not limited to: CT-011 in combination with Rituximab (trade namesRituxan, MabThera and Zytux) a chimeric monoclonal antibody against theprotein CD20, for example, each at 3 mg/kg; BMS-936558 (for example 1mg/kg) in combination with ipilimumab; for example at 3 mg/kg); orBMS-936558 (e.g. 1-10 mg/kg) in combination with a anHLA-A*0201-restricted multipeptide vaccine (Weber et al, 2013).

In certain embodiments, the agent is a p300 inhibitor, which formulasare listed in Table 1, i.e. C646(4-(4-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoicacid), C146 (4-hydroxy-3-(((2-(3odophenyl)benzo[d]oxazol-5-yl)imino)methyl)benzoic acid) or C375(2-chloro-4-(5-((2,4-dioxo-3-(2-oxo-2-(p-tolylamino)ethyl)thiazolidin-5-ylidene)methyl)furan-2-yl)benzoicacid). In particular, the p300 inhibitor is C646.

In certain embodiments, the adenosine receptor antagonist may be CGS15943 (9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine);the adenosine A1 receptor antagonist may be PSB 36(1-Butyl-8-(hexahydro-2,5-methanopentalen-3a(1H)-yl)-3,7-dihydro-3-(3-ydroxypropyl)-1H-purine-2,6-dione);the adenosine A2a receptor antagonist may be SCH58261(5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine),SYN 115(4-Hydroxy-N-[4-methoxy-7-(4-morpholinyl)-2-benzothiazolyl]-4-methyl-1-piperidinecarboxamide),FSPTP (also called SCH58261(5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ε]-1,2,4-triazolo[1,5-c]pyrimidine),SCH442416(2-(2-Furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine),or ZM241385 (also called tozadenant(4-Hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide);the adenosine A2b receptor antagonist may be PSB 603(8-{4-[4-(4-chlorophenyl)piperazine-1-sulfonyl]phenyl}-1-propyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione(Nakatsukasa et al, 2011)); and the A3 receptor antagonist may beMRS3777 (2-Phenoxy-6-(cyclohexylamino)purine hemioxalate).

In certain embodiments, the small molecule inhibitor of theindoleamine-2,3-dioxygenase pathway may be Indoximod(NSC-721782/NLG-9189 (1-Methyl-D-tryptophan), NewLink Genetics),INCB024360((4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine,Incyte) or NLG-919(1-Cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol), NewLinkGenetics).

The HIF-1 regulator may be M30,(5[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline) described inZheng et al. (Zheng et al, 2015).

In certain embodiments, the agent can be derived from a broad spectrumof antibiotics which targets gram-positive and gram-negative bacteria,and thereby facilitating immunomodulation of Tregs, e.g. vancomycinwhich targets gram-positive bacteria and has been shown to reduce Treglevels/activity (Brestoff & Artis, 2013; Smith et al, 2013).

As stated above, the active agent is administered by a dosage regimecomprising at least two courses of therapy, each course of therapycomprising in sequence a treatment session followed by an intervalsession. The dosage regime may be determined in a number of ways. Forexample, the level of immunosuppression may be calibrated to a desiredlevel for each patient who is being treated (personalized medicine), bymonitoring the level or activity of IFN-γ-producing leukocytes orproliferation rate of leukocytes in response to stimulationindividually, and adjusting the treatment session, the frequency ofadministration and the interval session empirically and personally asdetermined from the results of the monitoring.

Thus, the treatment session may comprise administering the active agentor pharmaceutical composition to the individual and the treatmentsession is maintained at least until the systemic presence or level ofIFN-γ-producing leukocytes, or the rate of proliferation of leukocytesin response to stimulation rises above a reference, the administering ispaused during the interval session, and the interval session ismaintained as long as the level is above the reference, wherein thereference is selected from (a) the level of systemic presence oractivity of IFN-γ-producing leukocytes, or the rate of proliferation ofleukocytes in response to stimulation, measured in the most recent bloodsample obtained from said individual before said administering; or (b)the level of systemic presence or activity of IFN-γ-producingleukocytes, or the rate of proliferation of leukocytes in response tostimulation, characteristic of a population of individuals afflictedwith a disease, disorder, condition or injury of the CNS.

The length of the treatment and interval sessions may be determined byphysicians in clinical trials directed to a certain patient populationand then applied consistently to this patient population, without theneed for monitoring the level of immunosuppression on a personal basis.

In certain embodiments, the treatment session comprises administeringthe active agent to the individual and the treatment session ismaintained at least until the systemic presence of the active agentreaches therapeutic levels, the administering is paused during theinterval session, and the interval session is maintained as long as thelevel is above about 95%, 90%, 80%, 70%, 60% or 50% of said therapeuticlevel. The term “therapeutic level” as used herein refers to generallyaccepted systemic levels of drugs used to block immune checkpoints inknown therapies, such as cancer therapy (see above).

In certain embodiments, the treatment session may be between 3 days andfour weeks long, for example between one and four weeks long.

In certain embodiments, the interval session may be between one week andsix months, for example between two weeks and six months long, inparticular between 3 weeks and six months long.

In the treatments session, the administration of the active agent orpharmaceutical composition may be repeated administration, for examplethe active agent or pharmaceutical composition may be administereddaily, or once every two, three, four, five or six days, once weekly,once every two weeks, once every three weeks or once every four weeks.These frequencies are applicable to any active agent, may be based oncommonly used practices in the art, and may finally be determined byphysicians in clinical trials. Alternatively, the frequency of therepeated administration in the treatment session could be adaptedaccording to the nature of the active agent, wherein for example, asmall molecule may be administered daily and an antibody may beadministered once every 3 days. It should be understood that when anagent is administered during a treatment session at a relatively lowfrequency, for example once per week during a treatment session of onemonth, or once per month during a treatment session of six months, thistreatment session is followed by a non-treatment interval session, thelength of which is longer than the period between the repeatedadministrations during the treatment session (i.e. longer than one weekor one month, respectively, in this example). The pause of one week orone month between the administrations during the treatment session inthis example is not considered an interval session.

The lengths of the treatment session and the interval session may beadjusted to the frequency of the administration such that, for example,a frequency of administering the active agent once every 3 days mayresult in a treatment session of 6 or 9 days and an interval sessionthat is commenced accordingly.

In any case, the dosage regimen, i.e. the length of the treatmentsession and the interval session, is calibrated such that the reductionin the level of immunosuppression, for example as measured by areduction in the level of systemic presence or activity of regulatory Tcells or the increase in the level of systemic presence or activity ofIFN-γ producing leukocytes in the individual, is transient.

The method, active agent or pharmaceutical composition according to thepresent invention may be for treating a disease, disorder or conditionof the CNS that is a neurodegenerative disease, disorder or conditionselected from Alzheimer's disease, amyotrophic lateral sclerosis,Parkinson's disease Huntington's disease, primary progressive multiplesclerosis; secondary progressive multiple sclerosis, corticobasaldegeneration, Rett syndrome, a retinal degeneration disorder selectedfrom the group consisting of age-related macular degeneration andretinitis pigmentosa; anterior ischemic optic neuropathy; glaucoma;uveitis; depression; trauma-associated stress or post-traumatic stressdisorder, frontotemporal dementia, Lewy body dementias, mild cognitiveimpairments, posterior cortical atrophy, primary progressive aphasia orprogressive supranuclear palsy. In certain embodiments, the condition ofthe CNS is aged-related dementia.

In certain embodiments, the condition of the CNS is Alzheimer's disease,amyotrophic lateral sclerosis, Parkinson's disease Huntington's disease.

The method, active agent and pharmaceutical composition according to thepresent invention may further be for treating an injury of the CNSselected from spinal cord injury, closed head injury, blunt trauma,penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebralischemia, optic nerve injury, myocardial infarction, organophosphatepoisoning and injury caused by tumor excision

As stated above, the inventors have found that the present inventionimproves the cognitive function in mice that emulates Alzheimer'sdisease. Thus, the method, active agent and pharmaceutical compositionmay be for use in improving CNS motor and/or cognitive function, forexample for use in alleviating age-associated loss of cognitivefunction, which may occur in individuals free of a diagnosed disease, aswell as in people suffering from neurodegenerative disease. Furthermore,the method, active agent and pharmaceutical composition may be for usein alleviating loss of cognitive function resulting from acute stress ortraumatic episode. The cognitive function mentioned herein above maycomprise learning, memory or both.

The term “CNS function” as used herein refers, inter alia, to receivingand processing sensory information, thinking, learning, memorizing,perceiving, producing and understanding language, controlling motorfunction and auditory and visual responses, maintaining balance andequilibrium, movement coordination, the conduction of sensoryinformation and controlling such autonomic functions as breathing, heartrate, and digestion.

The terms “cognition”, “cognitive function” and “cognitive performance”are used herein interchangeably and are related to any mental process orstate that involves but is not limited to learning, memory, creation ofimagery, thinking, awareness, reasoning, spatial ability, speech andlanguage skills, language acquisition and capacity for judgmentattention. Cognition is formed in multiple areas of the brain such ashippocampus, cortex and other brain structures. However, it is assumedthat long term memories are stored at least in part in the cortex and itis known that sensory information is acquired, consolidated andretrieved by a specific cortical structure, the gustatory cortex, whichresides within the insular cortex.

In humans, cognitive function may be measured by any know method, forexample and without limitation, by the clinical global impression ofchange scale (CIBIC-plus scale); the Mini Mental State Exam (MMSE); theNeuropsychiatric Inventory (NPI); the Clinical Dementia Rating Scale(CDR); the Cambridge Neuropsychological Test Automated Battery (CANTAB)or the Sandoz Clinical Assessment-Geriatric (SCAG). Cognitive functionmay also be measured indirectly using imaging techniques such asPositron Emission Tomography (PET), functional magnetic resonanceimaging (fMRI), Single Photon Emission Computed Tomography (SPECT), orany other imaging technique that allows one to measure brain function.

An improvement of one or more of the processes affecting the cognitionin a patient will signify an improvement of the cognitive function insaid patient, thus in certain embodiments improving cognition comprisesimproving learning, plasticity, and/or long term memory. The terms“improving” and “enhancing” may be used interchangeably.

The term “learning” relates to acquiring or gaining new, or modifyingand reinforcing, existing knowledge, behaviors, skills, values, orpreferences.

The term “plasticity” relates to synaptic plasticity, brain plasticityor neuroplasticity associated with the ability of the brain to changewith learning, and to change the already acquired memory. One measurableparameter reflecting plasticity is memory extinction.

The term “memory” relates to the process in which information isencoded, stored, and retrieved. Memory has three distinguishablecategories: sensory memory, short-term memory, and long-term memory.

The term “long term memory” is the ability to keep information for along or unlimited period of time. Long term memory comprises two majordivisions: explicit memory (declarative memory) and implicit memory(non-declarative memory). Long term memory is achieved by memoryconsolidation which is a category of processes that stabilize a memorytrace after its initial acquisition. Consolidation is distinguished intotwo specific processes, synaptic consolidation, which occurs within thefirst few hours after learning, and system consolidation, wherehippocampus-dependent memories become independent of the hippocampusover a period of weeks to years.

The embodiments above that describe different features of thepharmaceutical composition of the present invention are relevant alsofor the method of the invention, because the method employs the samepharmaceutical composition.

In yet another aspect, the present invention provides methods forreducing AP-plaque burden in a patient diagnosed with Alzheimer'sdisease, comprising administering to said patient an active agent orpharmaceutical composition as defined herein above that causes reductionof the level of systemic immunosuppression by release of a restraintimposed on the immune system by one or more immune checkpoints.

In still another aspect, the present invention provides a method forreducing hippocampal gliosis in a patient diagnosed with Alzheimer'sdisease, comprising administering to said patient an active agent orpharmaceutical composition as defined herein above that causes reductionof the level of systemic immunosuppression by release of a restraintimposed on the immune system by one or more immune checkpoints.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The carrier(s) mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof.

The following exemplification of carriers, modes of administration,dosage forms, etc., are listed as known possibilities from which thecarriers, modes of administration, dosage forms, etc., may be selectedfor use with the present invention. Those of ordinary skill in the artwill understand, however, that any given formulation and mode ofadministration selected should first be tested to determine that itachieves the desired results.

Methods of administration include, but are not limited to, parenteral,e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal(e.g., oral, intranasal, buccal, vaginal, rectal, intraocular),intrathecal, topical and intradermal routes. Administration can besystemic or local.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the active agent is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; and a glidant, such as colloidal silicondioxide.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compositions may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen free water, before use.

The compositions may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The determination of the doses of the active ingredient to be used forhuman use is based on commonly used practices in the art, and will befinally determined by physicians in clinical trials. An expectedapproximate equivalent dose for administration to a human can becalculated based on the in vivo experimental evidence disclosed hereinbelow, using known formulas (e.g. Reagan-Show et al. (2007) Dosetranslation from animal to human studies revisited. The FASEB Journal22:659-661). According to this paradigm, the adult human equivalent dose(mg/kg body weight) equals a dose given to a mouse (mg/kg body weight)multiplied with 0.081.

The invention will now be illustrated by the following non-limitingexamples.

Examples Materials and Methods

Animals.

5×FAD transgenic mice (Tg6799) that co-overexpress familial AD mutantforms of human APP (the Swedish mutation, K670N/M671L; the Floridamutation, 1716V; and the London mutation, V717I) and PS1 (M146L/L286V)transgenes under transcriptional control of the neuron-specific mouseThy-1 promoter (Oakley et al, 2006), and AD double transgenic B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/J mice (Borchelt et al, 1997) were purchasedfrom The Jackson Laboratory. Genotyping was performed by PCR analysis oftail DNA, as previously described (Oakley et al, 2006). Heterozygousmutant cx₃cr1^(GFP/+) mice (Jung et al, 2000)(B6.129P-cx3cr1^(tm1Litt)/J, in which one of the CX₃CR1 chemokinereceptor alleles was replaced with a gene encoding GFP) were used asdonors for BM chimeras. Foxp3.LuciDTR mice (Suffner et al, 2010) werebred with 5×FAD mice to enable conditional depletion of Foxp3⁺ Tregs.Animals were bred and maintained by the Animal Breeding Center of theWeizmann Institute of Science. All experiments detailed herein compliedwith the regulations formulated by the Institutional Animal Care and UseCommittee (IACUC) of the Weizmann Institute of Science.

RNA Purification, cDNA Synthesis, and Quantitative Real-Time PCRAnalysis.

Total RNA of the hippocampal dentate gyrus (DG) was extracted withReagent (Molecular Research Center) and purified from the lysates usingan RNeasy Kit (Qiagen). Total RNA of the choroid plexus was extractedusing an RNA MicroPrep Kit (Zymo Research). mRNA (1 μg) was convertedinto cDNA using a High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems). The expression of specific mRNAs was assayed usingfluorescence-based quantitative real-time PCR (RT-qPCR). RT-qPCRreactions were performed using Fast-SYBR PCR Master Mix (AppliedBiosystems). Quantification reactions were performed in triplicate foreach sample using the standard curve method. Peptidylprolyl isomerase A(ppia) was chosen as a reference (housekeeping) gene. The amplificationcycles were 95° C. for 5 s, 60° C. for 20 s, and 72° C. for 15 s. At theend of the assay, a melting curve was constructed to evaluate thespecificity of the reaction. For ifn-γ and ppia gene analysis, the cDNAwas pre-amplified in 14 PCR cycles with non-random PCR primers, therebyincreasing the sensitivity of the subsequent real-time PCR analysis,according to the manufacturer's protocol (PreAmp Master Mix Kit; AppliedBiosystems). mRNA expression was determined using TaqMan RT-qPCR,according to the manufacturer's instructions (Applied Biosystems). AllRT-qPCR reactions were performed and analyzed using StepOne softwareV2.2.2 (Applied Biosystems). The following TaqMan Assays-on-Demand™probes were used: Mm02342430_g1 (ppia) and Mm01168134_m1 (ifn-γ). Forall other genes examined, the following primers were used:

ppia forward (SEQ ID NO: 33) 5′-AGCATACAGGTCCTGGCATCTTGT-3′ and reverse(SEQ ID NO: 34) 5′-CAAAGACCACATGCTTGCCATCCA-3′; icam 1 forward(SEQ ID NO: 35) 5′-AGATCACATTCACGGTGCTGGCTA-3′ and reverse(SEQ ID NO: 36) 5′-AGCTTTGGGATGGTAGCTGGAAGA-3′; vcam 1 forward(SEQ ID NO: 37) 5′-TGTGAAGGGATTAACGAGGCTGGA-3′ and reverse(SEQ ID NO: 38) 5′-CCATGTTTCGGGCACATTTCCACA-3′; cxcl10 forward(SEQ ID NO: 39) 5′-AACTGCATCCATATCGATGAC-3′ and reverse (SEQ ID NO: 40)5′-GTGGCAATGATCTCAACAC-3′; ccl2 forward (SEQ ID NO: 41)5′-CATCCACGTGTTGGCTCA-3′ and reverse (SEQ ID NO: 42)5′-GATCATCTTGCTGGTGAATGAGT-3′; tnf-γ forward (SEQ ID NO: 43)5′-GCCTCTTCTCATTCCTGCTT-3′ reverse (SEQ ID NO: 44)CTCCTCCACTTGGTGGTTTG-3′; il-1β forward (SEQ ID NO: 45)5′-CCAAAAGATGAAGGGCTGCTT-3′ and reverse (SEQ ID NO: 46)5′-TGCTGCTGCGAGATTTGAAG-3′; il-12p40 forward (SEQ ID NO: 47)5′-GAAGTTCAACATCAAGAGCA-3′ and reverse (SEQ ID NO: 48)5′-CATAGTCCCTTTGGTCCAG-3′; il-10 forward (SEQ ID NO: 49)5′-TGAATTCCCTGGGTGAGAAGCTGA-3′ and reverse (SEQ ID NO: 50)5′-TGGCCTTGTAGACACCTTGGTCTT-3′; tgfβ2 forward (SEQ ID NO: 51)5′-AATTGCTGCCTTCGCCCTCTTTAC-3′ and reverse (SEQ ID NO: 52)5′-TGTACAGGCTGAGGACTTTGGTGT-3′; igf-1 forward (SEQ ID NO: 53)5′-CCGGACCAGAGACCCTTTG and reverse (SEQ ID NO: 54)5′-CCTGTGGGCTTGTTGAAGTAAAA-3′; bdnf forward (SEQ ID NO: 55)5′-GATGCTCAGCAGTCAAGTGCCTTT-3′ and reverse (SEQ ID NO: 56)5′-GACATGTTTGCGGCATCCAGGTAA-3′;

Immunohistochemistry.

Tissue processing and immunohistochemistry were performed on paraffinembedded sectioned mouse (6 μm thick) and human (10 μm thick) brains.For human ICAM-1 staining, primary mouse anti-ICAM (1:20 Abcam; ab2213)antibody was used. Slides were incubated for 10 min with 3% H2O2, and asecondary biotin-conjugated anti-mouse antibody was used, followed bybiotin/avidin amplification with Vectastain ABC kit (VectorLaboratories). Subsequently, 3,3′-diaminobenzidine (DAB substrate)(Zytomed kit) was applied; slides were dehydrated and mounted withxylene-based mounting solution. For tissue stainings, mice weretranscardially perfused with PBS prior to tissue excision and fixation.CP tissues were isolated under a dissecting microscope (Stemi DV4;Zeiss) from the lateral, third, and fourth ventricles of the brain. Forwhole mount CP staining, tissues were fixated with 2.5% paraformaldehyde(PFA) for 1 hour at 4° C., and subsequently transferred to PBScontaining 0.05% sodium azide. Prior to staining, the dissected tissueswere washed with PBS and blocked (20% horse serum, 0.3% Triton X-100,and PBS) for 1 h at room temperature. Whole mount staining with primaryantibodies (in PBS containing 2% horse serum and 0.3% Triton X-100), orsecondary antibodies, was performed for 1 h at room temperature. Eachstep was followed by three washes in PBS. The tissues were applied toslides, mounted with Immu-mount (9990402, from Thermo Scientific), andsealed with cover-slips. For staining of sectioned brains, two differenttissue preparation protocols (paraffin embedded or microtomedfree-floating sections) were applied, as previously described (Baruch etal, 2013; Kunis et al, 2013). The following primary antibodies wereused: mouse anti-Aβ (1:300, Covance, #SIG-39320); rabbit anti-GFP(1:100, MBL, #598); rat anti-CD68 (1:300, eBioscience, #14-0681); ratanti-ICAM-1 (1:200, Abcam, #AB2213); goat anti-GFP (1:100, Abcam,#ab6658); rabbit anti-IBA-1 (1:300, Wako, #019-19741); goat anti-IL-10(1:20, R&D systems, #μF519); rat anti-Foxp3 (1:20, eBioscience,#13-5773-80); rabbit anti-CD3 (1:500, Dako, #IS503); mouse anti-ZO-1,mouse anti-E-Cahedrin, and rabbit anti-Claudin-1 (all 1:100, Invitrogen,#33-9100, #33-4000, #51-9000); rabbit anti-GFAP (1:200, Dako, #Z0334).Secondary antibodies included: Cy2/Cy3/Cy5-conjugated donkeyanti-mouse/goat/rabbit/rat antibodies (1:200; all from JacksonImmunoresearch). The slides were exposed to Hoechst nuclear staining(1:4000; Invitrogen Probes) for 1 min. Two negative controls wereroutinely used in immunostaining procedures, staining with isotypecontrol antibody followed by secondary antibody, or staining withsecondary antibody alone. For Foxp3 intracellular staining, antigenretrieval from paraffin-embedded slides was performed using RetreivagenKit (#550524, #550527; BD Pharmingen™) Microscopic analysis, wasperformed using a fluorescence microscope (E800; Nikon) orlaser-scanning confocal microscope (Carl Zeiss, Inc.). The fluorescencemicroscope was equipped with a digital camera (DXM 1200F; Nikon), andwith either a 20×NA 0.50 or 40×NA 0.75 objective lens (Plan Fluor;Nikon). The confocal microscope was equipped with LSM 510 laser scanningcapacity (three lasers: Ar 488, HeNe 543, and HeNe 633). Recordings weremade on postfixed tissues using acquisition software (NIS-Elements, F3[Nikon] or LSM [Carl Zeiss, Inc.]). For quantification of stainingintensity, total cell and background staining was measured using ImageJsoftware (NIH), and intensity of specific staining was calculated, aspreviously described (Burgess et al, 2010). Images were cropped, merged,and optimized using Photoshop CS6 13.0 (Adobe), and were arranged usingIllustrator CS5 15.1 (Adobe).

Paraffin Embedded Sections of Human CP.

Human brain sections of young and aged postmortem non-CNS-diseaseindividuals, as well as AD patients, were obtained from the Oxford BrainBank (formerly known as the Thomas Willis Oxford Brain Collection(TWOBC)) with appropriate consent and Ethics Committee approval (TW220).The experiments involving these sections were approved by the WeizmannInstitute of Science Bioethics Committee.

Flow Cytometry, Sample Preparation and Analysis.

Mice were transcardially perfused with PBS, and tissues were treated aspreviously described (Baruch et al, 2013). Brains were dissected and thedifferent brain regions were removed under a dissecting microscope(Stemi DV4; Zeiss) in PBS, and tissues were dissociated using thegentleMACS™ dissociator (Miltenyi Biotec). Choroid plexus tissues wereisolated from the lateral, third and fourth ventricles of the brain,incubated at 37° C. for 45 min in PBS (with Ca²⁺/Mg²⁺) containing 400U/ml collagenase type IV (Worthington Biochemical Corporation), and thenmanually homogenized by pipetting. Spleens were mashed with the plungerof a syringe and treated with ACK (ammonium chloride potassium) lysingbuffer to remove erythrocytes. In all cases, samples were stainedaccording to the manufacturers' protocols. All samples were filteredthrough a 70 μm nylon mesh, and blocked with anti-Fc CD16/32 (1:100; BDBiosciences). For intracellular staining of IFN-γ, the cells wereincubated with para-methoxyamphetamine (10 ng/ml; Sigma-Aldrich) andionomycin (250 ng/ml; Sigma-Aldrich) for 6 h, and Brefeldin-A (10 μg/ml;Sigma-Aldrich) was added for the last 4 h. Intracellular labeling ofcytokines was done with BD Cytofix/Cytoperm™ Plusfixation/permeabilization kit (cat. no. 555028). For Treg staining, aneBioscience FoxP3 staining buffer set (cat. no. 00-5523-00) was used.The following fluorochrome-labeled monoclonal antibodies were purchasedfrom BD Pharmingen™, BioLegend, R&D Systems, or eBiosciences, and usedaccording to the manufacturers' protocols: PE or Alexa Fluor450-conjugated anti-CD4; PE-conjugated anti-CD25; PerCP-Cy5.5-conjugatedanti-CD45; FITC-conjugated anti-TCRβ; APC-conjugated anti-IFN-γ;APC-conjugated anti-FoxP3; Brilliant-violet-conjugated anti-CD45. Cellswere analyzed on an LSRII cytometer (BD Biosciences) using FlowJosoftware. In each experiment, relevant negative control groups, positivecontrols, and single stained samples for each tissue were used toidentify the populations of interest and to exclude other populations.

Preparation of BM Chimeras.

BM chimeras were prepared as previously described (Shechter et al, 2009;Shechter et al, 2013). In brief, gender-matched recipient mice weresubjected to lethal whole-body irradiation (950 rad) while shielding thehead (Shechter et al, 2009). The mice were then injected intravenouslywith 5×10⁶ BM cells from CX₃CR1^(GFP/+) donors. Mice were left for 8-10weeks after BM transplantation to enable reconstitution of thehematopoietic lineage, prior to their use in experiments. The percentageof chimerism was determined by FACS analysis of blood samples accordingto percentages of GFP expressing cells out of circulating monocytes(CD11b⁺). In this head-shielded model, an average of 60% chimerism wasachieved, and CNS-infiltrating GFP⁺ myeloid cells were verified to beCD45^(high)/CD11b^(high), representing monocyte-derived macrophages andnot microglia (Shechter et al, 2013).

Morris Water Maze.

Mice were given three trials per day, for 4 consecutive days, to learnto find a hidden platform located 1.5 cm below the water surface in apool (1.1 m in diameter). The water temperature was kept between 21-22°C. Water was made opaque with milk powder. Within the testing room, onlydistal visual shape and object cues were available to the mice to aid inlocation of the submerged platform. The escape latency, i.e., the timerequired to find and climb onto the platform, was recorded for up to 60s. Each mouse was allowed to remain on the platform for 15 s and wasthen removed from the maze to its home cage. If the mouse did not findthe platform within 60 s, it was manually placed on the platform andreturned to its home cage after 15 s. The inter-trial interval for eachmouse was 10 min. On day 5, the platform was removed, and mice weregiven a single trial lasting 60 s without available escape. On days 6and 7, the platform was placed in the quadrant opposite the originaltraining quadrant, and the mouse was retrained for three sessions eachday. Data were recorded using the EthoVision V7.1 automated trackingsystem (Noldus Information Technology). Statistical analysis wasperformed using analysis of variance (ANOVA) and the Bonferroni post-hoctest. All MWM testing was performed between 10 a.m. and 5 p.m. duringthe lights-off phase.

Radial Arm Water Maze.

The radial-arm water maze (RAWM) was used to test spatial learning andmemory, as was previously described in detail (Alamed et al, 2006).Briefly, six stainless steel inserts were placed in the tank, formingsix swim arms radiating from an open central area. The escape platformwas located at the end of one arm (the goal arm), 1.5 cm below the watersurface, in a pool 1.1 m in diameter. The water temperature was keptbetween 21-22° C. Water was made opaque with milk powder. Within thetesting room, only distal visual shape and object cues were available tothe mice to aid in location of the submerged platform. The goal armlocation remained constant for a given mouse. On day 1, mice weretrained for 15 trials (spaced over 3 h), with trials alternating betweena visible and hidden platform, and the last 4 trails with hiddenplatform only. On day 2, mice were trained for 15 trials with the hiddenplatform. Entry into an incorrect arm, or failure to select an armwithin 15 sec, was scored as an error. Spatial learning and memory weremeasured by counting the number of arm entry errors or the escapelatency of the mice on each trial. Training data were analyzed as themean errors or escape latency, for training blocks of three consecutivetrials.

GA Administration.

Each mouse was subcutaneously (s.c.) injected with a total dose of 100μg of GA (batch no. P53640; Teva Pharmaceutical Industries, Petah Tiqva,Israel) dissolved in 200 μl of PBS. Mice were either injected accordingto a weekly-GA regimen (Butovsky et al, 2006), or daily-GAadministration (FIG. 8 and FIG. 16). Mice were euthanized either 1 weekafter the last GA injection, or 1 month after treatment, as indicatedfor each experiment.

Conditional Ablation of Treg.

Diphtheria toxin (DTx; 8 ng/g body weight; Sigma) was injectedintraperitoneally (i.p.) daily for 4 consecutive days to Foxp3.LuciDTRmice (Suffner et al, 2010). The efficiency of DTx was confirmed by flowcytometry analysis of immune cells in the blood and spleen, achievingalmost complete (>99%) depletion of the GFP-expressing FoxP3⁺CD4⁺ Tregcells (FIG. 4).

P300 Inhibition.

Inhibition of p300 in mice was performed similarly to previouslydescribed (Liu et al, 2013). p300i (C646; Tocris Bioscience) wasdissolved in DMSO and injected i.p. daily (8.9 mg kg⁻¹ d⁻¹, i.p.) for 1week. Vehicle-treated mice were similarly injected with DMSO.

ATRA Treatment.

All-trans retinoic acid (ATRA) administration to mice was performedsimilarly to previously described (Walsh et al, 2014). ATRA (Sigma) wasdissolved in DMSO and injected i.p. (8 mg kg⁻¹ d⁻¹) every other day overthe course of 1 week. Vehicle-treated mice were similarly injected withDMSO.

Soluble Aβ (sAβ) Protein Isolation and Quantification.

Tissue homogenization and sAβ protein extraction was performed aspreviously described (Schmidt et al, 2005). Briefly, cerebral brainparenchyma was dissected and snap-frozen and kept at −80° C. untilhomogenization. Proteins were sequentially extracted from samples toobtain separate fractions containing proteins of differing solubility.Samples were homogenized in 10 volumes of ice-cold tissue homogenizationbuffer, containing 250 mM of sucrose, 20 mM of Tris base, 1 mM ofethylenediaminetetraacetic acid (EDTA), and 1 mM of ethylene glycoltetraacetic acid (pH 7.4), using a ground glass pestle in a Douncehomogenizer. After six strokes, the homogenate was mixed 1:1 with 0.4%diethylamine (DEA) in a 100-mM NaCl solution before an additional sixstrokes, and then centrifuged at 135,000 g at 4° C. for 45 min. Thesupernatant (DEA-soluble fraction containing extracellular and cytosolicproteins) was collected and neutralized with 10% of 0.5M of Tris-HCl (pH6.8). Aβ₁₋₄₀ and Aβ₁₋₄₂ were individually measured by enzyme-linkedimmunosorbent assay (ELISA) from the soluble fraction using commerciallyavailable kits (Biolegend; #SIG-38954 and #SIG-38956, respectively)according to the manufacturer instructions.

Aβ Plaque Quantitation.

From each brain, 6 μm coronal slices were collected, and eight sectionsper mouse, from four different pre-determined depths throughout theregion of interest (dentate gyrus or cerebral cortex) wereimmunostained. Histogram-based segmentation of positively stained pixelswas performed using the Image-Pro Plus software (Media Cybernetics,Bethesda, Md., USA). The segmentation algorithm was manually applied toeach image, in the dentate gyrus area or in the cortical layer V, andthe percentage of the area occupied by total Aβ immunostaining wasdetermined. Plaque numbers were quantified from the same 6 μm coronalbrain slices, and are presented as average number of plaques per brainregion. Prior to quantification, slices were coded to mask the identityof the experimental groups, and plaque burden was quantified by anobserver blinded to the identity of the groups.

Statistical Analysis.

The specific tests used to analyze each set of experiments are indicatedin the figure legends. Data were analyzed using a two-tailed Student's ttest to compare between two groups, one-way ANOVA was used to compareseveral groups, followed by the Newman-Keuls post-hoc procedure forpairwise comparison of groups after the null hypothesis was rejected(P<0.05). Data from behavioral tests were analyzed using two-wayrepeated-measures ANOVA, and Bonferroni post-hoc procedure was used forfollow-up pairwise comparison. Sample sizes were chosen with adequatestatistical power based on the literature and past experience, and micewere allocated to experimental groups according to age, gender, andgenotype. Investigators were blinded to the identity of the groupsduring experiments and outcome assessment. All inclusion and exclusioncriteria were pre-established according to the IACUC guidelines. Resultsare presented as means±s.e.m. In the graphs, γ-axis error bars represents.e.m. Statistical calculations were performed using the GraphPad Prismsoftware (GraphPad Software, San Diego, Calif.).

Example 1 Choroid Plexus (CP) Gateway Activity Along Disease Progressionin the Mouse Model of AD

We first examined CP activity along disease progression in the 5×FADtransgenic mouse model of AD (AD-Tg); these mice co-express fivemutations associated with familial AD and develop cerebral Aβ pathologyand gliosis as early as 2 months of age (Oakley et al, 2006). We foundthat along the progressive stages of disease pathology, the CP of AD-Tgmice, compared to age-matched wild-type (WT) controls, expressedsignificantly lower levels of leukocyte homing and traffickingdeterminants, including icam1, vcam1, cxcl10, and ccl2 (FIG. 1A), shownto be upregulated by the CP in response to acute CNS damage, and neededfor transepithelial migration of leukocytes (Kunis et al, 2013; Shechteret al, 2013). Immunohistochemical staining for the integrin ligand,ICAM-1, confirmed its reduced expression by the CP epithelium of AD-Tgmice (FIG. 1b ). In addition, staining for ICAM-1 in human postmortembrains, showed its age-associated reduction in the CP epithelium, inline with our previous observations (Baruch et al, 2014), andquantitative assessment of this effect revealed further decline in ADpatients compared to aged individuals without CNS disease (FIG. 2A).Since the induction of leukocyte trafficking determinants by the CP isdependent on epithelial interferon (IFN)-γ signaling (Kunis et al,2013), we next tested whether the observed effects could reflect loss ofIFN-γ availability at the CP. Examining the CP of 5×FAD AD-Tg mice usingflow cytometry intracellular staining, revealed significantly lowernumbers of IFN-γ-producing cells in this compartment (FIG. 2B), andquantitative real-time PCR (RT-qPCR) analysis confirmed lower mRNAexpression levels of ifn-γ at the CP of AD-Tg mice compared toage-matched WT controls (FIG. 2C).

Example 2 The Functional Relationships Between Treg-Mediated SystemicImmune Suppression, CP Gateway Activity, and AD Pathology

Regulatory T cells (Tregs) play a pivotal role in suppressing systemiceffector immune responses (Sakaguchi et al, 2008). We envisioned thatTreg-mediated systemic immune suppression affects IFN-γ availability atthe CP, and therefore focused on the involvement of Tregs in ADpathology. In line with previous reports of elevated Treg levels andsuppressive activities in AD patients (Rosenkranz et al, 2007; Saresellaet al, 2010; Torres et al, 2013), evaluating Foxp3+ Treg frequencies insplenocytes of 5×FAD AD-Tg mice, relative to their age-matched WTlittermates, revealed their elevated levels along disease progression(FIG. 3A, B). To study the functional relationships betweenTreg-mediated systemic immune suppression, CP gateway activity, and ADpathology, we crossbred 5×FAD AD-Tg mice with Foxp3-diphtheria toxinreceptor (DTR⁺) mice, enabling transient conditional in vivo depletionof Foxp3⁺ Tregs in AD-Tg/DTR⁺ mice by administration of diphtheria toxin(DTx) (FIG. 4A). Transient depletion of Tregs resulted in elevated mRNAexpression of leukocyte trafficking molecules by the CP of AD-Tg/DTR⁺mice relative to DTx-treated AD-Tg/DTR⁻ littermates (FIG. 5A). Analysisof the long-term effect of the transient Treg depletion on the brainparenchyma (3 weeks later), revealed immune cell accumulation in thebrain, including elevated numbers of CD45^(high)/CD11b^(high) myeloidcells, representing infiltrating mo-MΦ (Shechter et al, 2013), and CD4⁺T cells (FIG. 5B). In addition, the short and transient depletion ofTregs resulted in a marked enrichment of Foxp3⁺ Tregs among the CD4⁺ Tcells that accumulated within the brain, as assessed by flow cytometry(FIG. 5C, D). RT-qPCR analysis of the hippocampus showed increasedexpression of foxp3 and il10 mRNA (FIG. 5E).

We next examined whether the short-term depletion of Tregs, which wasfollowed by accumulation of immunoregulatory cells in sites of brainpathology, led to a long-term effect on brain function. We observedreduction in hippocampal gliosis (FIG. 5F), and reduced mRNA expressionlevels of pro-inflammatory cytokines, such as il-12p40 and tnf-α (FIG.5G). Moreover, cerebral Aβ plaque burden in the hippocampal dentategyrus, and the cerebral cortex (5^(th) layer), two brain regionsexhibiting robust Aβ plaque pathology in 5×FAD AD-Tg mice (Oakley et al,2006), was reduced (FIG. 6A, B). Evaluating the effect on cognitivefunction, using the Morris water maze (MWM) test, revealed a significantimprovement in spatial learning and memory in AD-Tg/DTR⁺ mice followingthe Treg depletion, relative to DTx-treated AD-Tg/DTR⁻ aged matchedmice, reaching performance similar to that of WT mice (FIG. 6C-E). Takentogether, these data demonstrated that transiently breakingTreg-mediated systemic immune suppression in AD-Tg mice resulted inaccumulation of inflammation-resolving cells, including mo-MΦ and Tregs,in the brain, and was followed by resolution of the neuroinflammatoryresponse, clearance of AP, and reversal of cognitive decline.

Example 3 Weekly Administration of Copolymer-1 Reduces Treg-MediatedSystemic Immune Suppression, Improves CP Gateway Activity, and MitigatesAD Pathology

To further substantiate the causal nature of the inverse relationshipbetween systemic immune suppression, CP function and AD pathology, wenext made use of the immunomodulatory compound, Glatiramer acetate (GA;also known as Copolymer-1, or Copaxone®), which in a weeklyadministration regimen was found to have a therapeutic effect in theAPP/PS1 mouse model of AD (Butovsky et al, 2006); this effect wasfunctionally associated with mo-MΦ recruitment to cerebral sites ofdisease pathology (Butovsky et al, 2007). Here, we first examinedwhether the CP in APP/PS1 AD-Tg mice, similarly to our observation in5×FAD AD-Tg mice, is also deficient with respect to IFN-γ expressionlevels. We found that in APP/PS1 AD-Tg mice, IFN-γ levels at the CP werereduced relative to age-matched WT controls (FIG. 7A). These resultsencouraged us to test whether the therapeutic effect of weekly-GA inAPP/PS1 mice (Butovsky et al, 2006), could be reproduced in 5×FAD AD-Tgmice, and if so, whether it would affect systemic Tregs, and activationof the CP for mo-MΦ trafficking. We therefore treated 5×FAD AD-Tg micewith a weekly administration regimen of GA over a period of 4 weeks(henceforth, “weekly-GA”; schematically depicted in FIG. 8A). We foundthat 5×FAD AD-Tg mice treated with weekly-GA, showed reducedneuroinflammation (FIG. 8B-D), and improved cognitive performance, whichlasted up to 2 months after the treatment (FIG. 8E-I). Examining by flowcytometry the effect of weekly-GA on systemic immunity and on the CP, wefound reduced splenocyte Foxp3⁺ Treg levels (FIG. 9A), and an increasein IFN-γ-producing cells at the CP of the treated 5×FAD AD-Tg mice,reaching similar levels as those observed in WT controls (FIG. 9B). Theelevated level of IFN-γ-expressing cells at the CP in the weekly-GAtreated mice, was accompanied by upregulated epithelial expression ofleukocyte trafficking molecules (FIG. 9C).

To detect infiltrating mo-MΦ entry to the CNS, we used 5×FADAD-Tg/CX₃CR1^(GFP/+) bone marrow (BM) chimeric mice (prepared using headprotection), allowing the visualization of circulating (greenfluorescent protein (GFP)⁺ labeled) myeloid cells (Shechter et al, 2009;Shechter et al, 2013). We found increased homing of GFP⁺ mo-MΦ to the CPand to the adjacent ventricular spaces following weekly-GA treatment, ascompared to vehicle-treated AD-Tg/CX₃CR1^(GFP/+) controls (FIG. 9D-E).Immunohistochemistry of the brain parenchyma revealed the presence ofGFP⁺ mo-MΦ accumulation at sites of cerebral plaque formation (FIG. 9F),and quantification of infiltrating myeloid cells, by flow cytometryanalysis of the hippocampus in AD-Tg non-chimeric mice, showed increasednumbers of CD11b^(high)CD45^(high)-expressing cells (FIG. 9G, H).Together, these results substantiated the functional linkage betweenmo-MΦ recruitment to sites of AD pathology, reduction of systemic Treglevels and IFN-γ-dependent activation of the CP.

Example 4 Interference with Treg Activity Using a Small Molecule HistoneAcetyltransferase Inhibitor

The findings above, which suggested that Treg-mediated systemic immunesuppression interferes with the ability to fight AD pathology, arereminiscent of the function attributed to Tregs in cancer immunotherapy,in which these cells hinder the ability of the immune system to mount aneffective anti-tumor response (Bos & Rudensky, 2012; Nishikawa &Sakaguchi, 2010). Therefore, we considered that a treatment thatdirectly interferes with Foxp3⁺ Treg cell activity might be advantageousin AD. We tested p300i (C646 (Bowers et al, 2010)), a nonpeptidicinhibitor of p300, a histone acetyltransferase that regulates Tregfunction (Liu et al, 2013); this inhibitor was shown to affect Tregsuppressive activities while leaving protective T effector cellresponses intact (Liu et al, 2013). We found that mice treated withp300i, compared to vehicle (DMSO) treated controls, showed elevatedlevels of systemic IFN-γ-expressing cells in the spleen (FIG. 10A), aswell as in the CP (FIG. 10B). We next treated AD-Tg mice with eitherp300i or vehicle over the course of 1 week, and examined them 3 weekslater for cerebral Aβ plaque burden. Immunohistochemical analysisrevealed a significant reduction in cerebral Aβ plaque load in the p300itreated AD-Tg mice (FIG. 10C-E). We also tested whether the effect onplaque pathology following one course of treatment would last beyond the3 weeks, and if so, whether additional courses of treatment wouldcontribute to a long-lasting effect. We therefore compared AD-Tg micethat received a single course of p300i treatment and were examined 2month later, to an age-matched group that received two courses oftreatments during this period, with a 1-month interval in between(schematically depicted in FIG. 10F). We found that the reduction ofcerebral plaque load was evident even two months after a single courseof treatment, but was stronger in mice that received two courses oftreatments with a 1-month interval in between (FIG. 10G). Since impairedsynaptic plasticity and memory in AD is associated with elevatedcerebral levels of soluble Aβ₁₋₄₀/Aβ₁₋₄₂ (sAβ) levels (Shankar et al,2008), we also measured sAβ₃ levels following a single or repeatedcycles of p300i treatment. Again, we found that both one and two courses(with an interval of 1 month in between) were effective in reducingcerebral sAβ₃, yet this effect was stronger following repeated courseswith respect to the effect on sAβ₁₋₄₂ (FIG. 10H). These resultsindicated that while a single short-term course of treatment iseffective, repeated courses of treatments would be advantageous tomaintain a long-lasting therapeutic effect, similar to our observationsfollowing weekly-GA treatment.

Example 5 Therapeutic Potential of PD-1 Immune Checkpoint Blockade inAlzheimer's Disease

We first tested whether targeting the PD-1 inhibitory pathway couldaffect IFN-γ-associated systemic immunity in 5×FAD AD transgenic (AD-Tg)mice, which co-expresses five mutations associated with familial AD(Oakley et al, 2006). AD-Tg mice at the age of 10 months, a time pointat which cerebral pathology is advanced, were administrated with twointraperitoneal (i.p.) injections of either blocking antibodies directedat PD-1 (anti-PD-1) or IgG control antibodies, on days 1 and 4, and thenexamined on day 7. Flow cytometry analysis revealed that blockade of thePD-1 pathway resulted in elevated frequencies of IFN-γ-producing CD4⁺ Tsplenocytes (FIG. 11A, B).

TABLE 2 GO annotation, related to FIG. 11 FDR GO term DescriptionP-value q-value GO:0034341 response to interferon-gamma 2.13E−142.30E−10 GO:0048002 antigen processing and presentation 3.05E−101.65E−06 of peptide antigen GO:0019886 antigen processing andpresentation 4.11E−10 1.48E−06 of exogenous peptide antigen via MHCclass II GO:0002478 antigen processing and presentation 5.26E−101.42E−06 of exogenous peptide antigen GO:0034097 response to cytokine5.67E−10 1.22E−06 GO:0002504 antigen processing and presentation1.04E−09 1.87E−06 of peptide or polysaccharide antigen via MHC class IIGO:0002495 antigen processing and presentation 1.04E−09 1.60E−06 ofpeptide antigen via MHC class II GO:0019884 antigen processing andpresentation 5.82E−09 7.86E−06 of exogenous antigen GO:0019882 antigenprocessing and presentation 1.43E−07 1.71E−04 GO:0035456 response tointerferon-beta 6.67E−07 7.20E−04 GO:0006955 immune response 1.07E−061.05E−03 GO:0002819 regulation of adaptive 1.92E−06 1.73E−03 immuneresponse GO:0071345 cellular response to 2.21E−06 1.84E−03 cytokinestimulus GO:0071346 cellular response to 2.21E−06 1.71E−03interferon-gamma Gene ontology terms enriched in the CP of AD-Tg micetreated with anti-PD-1, when compared to IgG treated and untreated AD-Tgcontrols. Log 10 values of all RNA sequences of the CP were rankedaccording to their differential expression levels and analyzed.

We next examined whether this systemic immune response affected the CPactivity. Genome wide RNA-sequencing of the CP (Not shown; the fullanalysis will be disclosed in a report by the present inventors havingthe title of Example 5 and it can be obtained from the inventors uponrequest) showed an expression profile associated with response to IFN-γ(FIG. 11D and Table 2), and real-time quantitative PCR (RT-qPCR)verified elevated IFN-γ mRNA levels at the CP, when compared toIgG-treated or untreated AD-Tg controls (FIG. 11C). These findingsconfirmed a systemic, and CP tissue-specific, IFN-γ immune responsefollowing PD-1 blockade, and encouraged us to next test the effect ondisease pathology.

To examine the functional impact of PD-1 blockade on AD pathology, wetreated 10-month old AD-Tg mice with either anti-PD-1 or IgG controlantibodies, and evaluated the effect on spatial learning and memoryperformance, using the radial arm water maze (RAWM) task.

One month following treatment (two i.p. injections with 3-day interval),anti-PD1 treated AD-Tg mice exhibited a significant improvement incognitive function relative to IgG-treated or untreated age-matchedcontrols, reaching cognitive levels similar to that of age-matched WTmice (FIG. 12A). We next tested whether the benefit of PD-1 blockade oncognitive performance in AD-Tg mice would last beyond 1 month, andwhether additional therapeutic sessions would be advantageous. Wetreated AD-Tg mice with anti-PD-1 at the age of 10 months (“1 session”)or at both 10 and 11 months of age (“2 sessions”), and examined theoutcome on cognitive performance at the age of 12 months (schematicallydepicted in FIG. 12B). Control groups included WT mice, untreated AD-Tgmice, and AD-Tg mice that received two sessions of IgG treatment. Wefound that while a single session of anti-PD-1 administration had abeneficial effect on spatial learning and memory 1 month following thetreatment (FIG. 12A), no significant effect could be detected in micethat received a single session of treatment and were tested 2 monthslater (FIG. 12B). In contrast, AD-Tg mice that received two sessions ofanti-PD-1, at a 1-month interval, displayed cognitive performancesimilar to that of WT mice, at the end of the 2-month timeframe (FIG.12B).

Finally, we examined whether PD-1 blockade affected AD pathology asmanifested by cerebral Aβ plaque load and gliosis. Brains of AD-Tg micethat received anti-PD-1 or IgG in either one or two sessions wereexamined by immunohistochemistry for Aβ and glial fibrillary acidprotein (GFAP). We found that cerebral Aβ plaque burden was reduced inthe hippocampal dentate gyrus (FIG. 13A, B), and the cerebral cortex(5th layer) (FIG. 13A, C), two brain regions exhibiting robust Aβ plaquepathology in 5×FAD mice (Oakley et al, 2006). The effect on Aβ clearancewas evident following a single session of anti-PD-1 administration, andwas more robust following two sessions. Quantitative analysis of GFAPimmunostaining showed reduced hippocampal astrogliosis in both AD-Tgmice treated with 1 session, and those treated with 2 sessions of PD-1blockade, relative to IgG-treated controls (FIG. 13A, D).

Example 6 Therapeutic Potential of PD-1 in Combination with CTLA4 ImmuneCheckpoint Blockade in Alzheimer's Disease

At 10 months of age, 5×FAD Alzheimer's' disease (AD) transgenic (Tg)mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14; #BE0146;Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4 (InVivoMAbanti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG(IgG2a, #BE0089 or Polyclonal Syrian Hamster IgG, #BE0087; BioxcellLifesciences Pvt. LTD.) antibodies, on day 1 and day 4 of theexperiment, and are examined 3 weeks after for their cognitiveperformance by radial arm water maze (RAWM) spatial learning and memorytask, as described above.

Some mice receive an additional treatment session with an intervalsession of 3 weeks. Control groups are either treated with IgG oruntreated, and all groups of mice are tested for their cognitiveperformance 3 weeks later.

It is expected that the mice treated with the combination of antibodiesdisplay significant cognitive improvement in comparison to IgG-treatedand untreated AD-Tg mice as well as a significant reduction of cerebralplaque load.

Example 7 Therapeutic Potential of Immune Checkpoint Blockade Approch inPTSD Pathology

Severely stressful conditions or chronic stress can lead toposttraumatic stress disorder (PTSD) and depression. We adopted aphysiological PTSD-like animal model in which the mice exhibithypervigilant behaviour, impaired attention, increased risk assessment,and poor sleep (Lebow et al, 2012). In this experimental model of PTSDinduction, mice are habituated for 10 days to a reverse day/night cycle,inflicted with two episodes of electrical shocks (the trauma and thetrigger), referred to as a “PTSD induction”, and evaluated at differenttime points subsequent to trauma. Following the traumatic event mice areinjected with said compound which blocks immune checkpoints. The miceare treated according to one of the following regimens:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks; or    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks;    -   Mice are treated with any of the other active agents disclosed        herein, on day 1 and day 4 of the experiment, and examined after        an interval session of two weeks.

Some mice receive an additional treatment session with an appropriateinterval session.

It is expected that mice that receive the treatment do not displayanxiety behavior associated with PTSD in this experimental model, asassessed by time spent exploring and risk assessing in dark/light mazeor the other behavioral tasks described in (Lebow et al, 2012).

Example 8 Therapeutic Potential of Immune Checkpoint Blockade Approachin Parkinson's Disease Pathology

Parkinson disease (PD) transgenic (Tg) mice are used in theseexperiment. The mice are treated at the progressive stages of diseaseaccording to one of the following regimens:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks;    -   Mice are treated with any of the other active agents disclosed        herein, on day 1 and day 4 of the experiment, and examined after        an interval session of two weeks.

Some mice receive an additional treatment session with an appropriateinterval session (about 3 weeks to one month).

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod.

It is expected that PD-Tg mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. PD-Tg mice whichreceive two courses of therapy, and examined after an appropriateinterval session are expected to show a long-lasting therapeutic effect.To maintain this therapeutic effect mice are subjected to an activesession of treatment with an appropriate interval session between eachsession.

Example 9 Therapeutic Potential of PD-1 in Combination with CTLA4 ImmuneCheckpoint Blockade in Huntington's Disease Pathology

The model used in these experiments may be the Huntington's disease (HD)R_(6/2) transgenic mice (Tg) test system. R6/2 transgenic mice overexpress the mutated human huntingtin gene that includes the insertion ofmultiple CAG repeats mice at the progressive stages of disease. Thesemice show progressive behavioral-motor deficits starting as early as 5-6weeks of age, and leading to premature death at 10-13 weeks. Thesymptoms include low body weight, clasping, tremor and convulsions.

The mice are treated according to one of the following regimens whenthey are 45 days old:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks.    -   Mice are treated with any of the other active agents disclosed        herein, on day 1 and day 4 of the experiment, and examined after        an interval session of two weeks.

Some mice receive an additional treatment session with an appropriateinterval session (about 3 weeks to one month).

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod.

It is expected that HD-Tg mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. HD-Tg mice whichreceive which receive two courses of therapy, and examined after anappropriate interval session are expected to show a long-lastingtherapeutic effect. To maintain this therapeutic effect mice aresubjected to an active session of treatment with an appropriate intervalsession between each session.

Example 10 Therapeutic Potential of Immune Checkpoint Blockade Approachin Amyotrophic Lateral Sclerosis Pathology

The model used in this experiment may be the transgenic miceoverexpressing the defective human mutant SOD1 allele containing theGly93→Ala (G93A) gene (B6SJL-TgN (SOD1-G93A)1Gur (herein “ALS mice”).This model develop motor neuron disease and thus constitute an acceptedanimal model for testing ALS.

The mice are treated according to one of the following regimens whenthey are 75 days old:

-   -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG (IgG2a;        #BE0089; Bioxcell Lifesciences Pvt. LTD.) antibodies, on day 1        and day 4 following the traumatic event, and examined after an        additional interval session of two weeks;    -   Mice are injected i.p. with either 250 μg of anti-PD1 (RMP1-14;        #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA4        (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt.        LTD.) or control IgG (IgG2a, #BE0089 or Polyclonal Syrian        Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.)        antibodies, on day 1 and day 4 of the experiment, and examined        after an interval session of two weeks.    -   Mice are treated with any of the other active agents disclosed        herein, on day 1 and day 4 of the experiment, and examined after        an interval session of two weeks.

Some mice receive an additional treatment session with an appropriateinterval session (about 3 weeks to month).

Motor neurological functions are evaluated using for example the rotarodperformance test, which assesses the capacity of the mice to stay on arotating rod, or mice are allowed to grasp and hold onto a vertical wire(2 mm in diameter) with a small loop at the lower end. A vertical wireallows mice to use both fore- and hindlimbs to grab onto the wire. Thewire is maintained in a vertically oriented circular motion (the circleradius was 10 cm) at 24 rpm. The time that the mouse is able to hangonto the wire is recorded with a timer.

It is expected that ALS mice treated with one treatment session showsignificant improved motor performance, compared to IgG-treated orvehicle treated control group, or untreated group. ALS mice whichreceive which receive two courses of therapy, and examined after anappropriate interval session are expected to show a long-lastingtherapeutic effect. To maintain this therapeutic effect mice aresubjected to an active session of treatment with an appropriate intervalsession between each session.

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The invention claimed is:
 1. A method of treating an Alzheimer'sDisease, the method comprising administering to an individual in needthereof a composition comprising an anti-programmed death ligand 1(PDL1) antibody, wherein the composition is administered by a dosageregime comprising at least two courses of therapy, each course oftherapy comprising in sequence a treatment session where the compositionis administered to the individual followed by a non-treatment periodwhere the composition is not administered to the individual, whereinadministration of the composition transiently reduces levels of systemicimmunosuppression and increases choroid plexus gateway activity infacilitating selective recruitment of immune cells into the centralnervous system, thereby treating the individual.
 2. The method accordingto claim 1, wherein the administration of the composition during thetreatment session is a single administration.
 3. The method according toclaim 1, wherein the administration of the composition during thetreatment session is a repeated administration.
 4. The method accordingto claim 3, wherein the repeated administration occurs once every two,three, four, five or six days.
 5. The method according to claim 3,wherein the repeated administration occurs once weekly.
 6. The methodaccording to claim 3, wherein the repeated administration occurs onceevery four weeks.
 7. The method according to claim 1, wherein thetreatment session is from 3 days to four weeks.
 8. The method accordingto claim 7, wherein the treatment session is from one week to fourweeks.
 9. The method according to claim 1, wherein the non-treatmentperiod is longer than the treatment session.
 10. The method according toclaim 9, wherein the non-treatment period is from one week to sixmonths.
 11. The method according to claim 10, wherein the non-treatmentperiod is from two weeks to six months.
 12. The method according toclaim 1, wherein the anti-PDL1 antibody is a neutralizing anti-PDL1antibody.
 13. The method according to claim 1, wherein the transientreduction in the level of systemic immunosuppression is associated withan increase in a systemic presence or activity of IFNγ-producingleukocytes and/or an increase in a systemic presence or activity of anIFNγ cytokine.
 14. The method according to claim 1, wherein thetransient reduction in the level of systemic immunosuppression isassociated with an increase in a systemic presence or activity ofeffector T cells.
 15. The method according to claim 1, wherein thetransient reduction in the level of systemic immunosuppression isassociated with a decrease in a systemic presence or activity ofregulatory T cells and/or a decrease in a systemic presence of an IL-10cytokine.
 16. The method according to claim 1, wherein the transientreduction in the level of systemic immunosuppression is associated witha decrease in a systemic presence or myeloid-derived suppressor cells(MDSCs).
 17. The method according to claim 1, wherein the transientreduction in the level of systemic immunosuppression occurs by releaseof a restraint imposed on the immune system by one or more immunecheckpoints.
 18. The method according to claim 17, whereinadministration of the composition blocks the one or more immunecheckpoints, thereby causing the transient reduction in the level ofsystemic immunosuppression.
 19. The method according to claim 18,wherein the one or more immune checkpoints includes PD1-PDL1.
 20. Themethod according to claim 1, wherein the administration of thecomposition during the treatment session is maintained at least until asystemic presence or activity of IFNγ-producing leukocytes and/or anIFNγ cytokine rises above a reference, at which point the administrationis stopped, and the non-treatment period is maintained as long as thesystemic presence or activity of IFNγ-producing leukocytes and/or anIFNγ cytokine is above the reference, wherein the reference includes a)a level of a systemic presence or activity of IFNγ-producing leukocytesand/or an IFNγ cytokine measured in the most recent blood sampleobtained from the individual before the administering; or b) a level ofa systemic presence or activity of IFNγ-producing leukocytes and/or anIFNγ cytokine characteristic of a population of individuals afflictedwith the Alzheimer's Disease.
 21. The method according to claim 1,wherein a cerebral level of soluble amyloid beta peptide is reduced inthe individual, a cerebral amyloid beta (Aβ) plaque burden is reduced orcleared in the individual, a hippocampal gliosis is reduced in theindividual, a cerebral level of a pro-inflammatory cytokine is reducedin the individual, a brain inflammation is decreased in the individualand/or a cognitive function is improved in the individual.
 22. Themethod according to claim 21, wherein the improved cognitive function islearning, memory, creation of imagery, plasticity, thinking, awareness,reasoning, spatial ability, speech and language skills, languageacquisition, capacity for judgment attention or any combination thereof.23. The method according to claim 1, wherein the immune cells includemonocytes, macrophages, or T cells.
 24. The method according to claim23, wherein the T cells include regulatory T cells.
 25. A method oftreating an Alzheimer's Disease, the method comprising administering toan individual in need thereof a composition comprising ananti-programmed death ligand 1 (PDL1) antibody as the only activeingredient, wherein the composition is administered by a dosage regimecomprising at least two courses of therapy, each course of therapycomprising in sequence a treatment session where the composition isadministered to the individual followed by a non-treatment period wherethe composition is not administered to the individual, whereinadministration of the composition transiently reduces levels of systemicimmunosuppression and increases choroid plexus gateway activity infacilitating selective recruitment of immune cells into the centralnervous system, thereby treating the individual.
 26. The methodaccording to claim 25, wherein the non-treatment period is longer thanthe treatment session.
 27. The method according to claim 26, wherein thenon-treatment period is from one week to six months.
 28. The methodaccording to claim 27, wherein the non-treatment period is from twoweeks to six months.
 29. The method according to claim 26, wherein thetreatment session is from 3 days to four weeks.
 30. The method accordingto claim 28, wherein the non-treatment period is from three weeks to sixmonths.
 31. The method according to claim 29, wherein the treatmentsession is from one week to four weeks.
 32. The method according toclaim 11, wherein the non-treatment period is from three weeks to sixmonths.